WO2006112451A1 - Anti-cancer agent comprising protein c inhibitor - Google Patents

Anti-cancer agent comprising protein c inhibitor Download PDF

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Publication number
WO2006112451A1
WO2006112451A1 PCT/JP2006/308095 JP2006308095W WO2006112451A1 WO 2006112451 A1 WO2006112451 A1 WO 2006112451A1 JP 2006308095 W JP2006308095 W JP 2006308095W WO 2006112451 A1 WO2006112451 A1 WO 2006112451A1
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Prior art keywords
pci
mda
cancer
cells
protein
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PCT/JP2006/308095
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French (fr)
Japanese (ja)
Inventor
Koji Suzuki
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Mie University
Chugai Seiyaku Kabushiki Kaisha
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Application filed by Mie University, Chugai Seiyaku Kabushiki Kaisha filed Critical Mie University
Priority to US11/911,754 priority Critical patent/US8106002B2/en
Priority to EP06732024A priority patent/EP1894574A4/en
Priority to JP2007528151A priority patent/JPWO2006112451A1/en
Publication of WO2006112451A1 publication Critical patent/WO2006112451A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the present invention relates to an anticancer agent. More specifically, the present invention relates to an anticancer agent containing a protein C inhibitor, a method for treatment with an anticancer agent containing a protein c inhibitor, and a method for producing an anticancer agent containing a protein c inhibitor.
  • cancer is an important disease that dominates the leading cause of death, especially in many countries.
  • the annual number of deaths from cancer exceeds 300,000. This figure is approximately double the number of deaths from heart disease. Therefore, provision of cancer treatment technology is an important research subject.
  • Physiochemical treatment includes radiation therapy, particle beam (charged heavy particle beam) treatment, or hyperthermia.
  • Many chemotherapeutic agents used for drug treatment have also been put into practical use.
  • the therapeutic effects of new approaches such as vaccine therapy or cellular immunotherapy are being clarified.
  • providing cancer treatment methods remains an important research issue. In particular, if a technique for preventing cancer malignancy or a method for treating malignant cancer is provided, it will greatly contribute to cancer medical treatment.
  • cancer malignancy In general, the mechanism of cancer malignancy can be explained by the proliferation, invasion, and metastasis of cancer cells. In other words, when a cancer cell that has proliferated metastasizes by invasion, the cancer is considered malignant. Cancer growth is usually accompanied by angiogenesis. Therefore, angiogenesis is also an important mechanism that constitutes malignant cancer.
  • cancer cells that have proliferated in the primary lesion are detached from the primary lesion.
  • the detached cancer cells move by lymph fluid and blood flow and invade other tissues. Metastasis is established when the infiltrated cancer cells begin to grow again.
  • Cancer cell growth requires angiogenesis.
  • blood from the primary lesion Angiogenesis also has an important role in the removal of cancer cells into the flow. It is thought that malignant cancer can be prevented if any of these mechanisms can be prevented.
  • cell growth, infiltration, metastasis, and angiogenesis are important therapeutic targets in the treatment of cancer, especially malignant cancer.
  • Epithelial cells maintain a strong sheet-like structure by intercellular adhesion via E-cadherin. Therefore, the movement of epithelial cells is restricted. In malignant cancers, the adhesion between cells is weak, and it is thought that it is easy to leave the primary lesion.
  • ECM extracellular matrix
  • MMP which is a protease
  • MMP-1 and MMP-3 are known to be activated by plasmin.
  • Plasmin acquires protease activity through the activity of plasminogen.
  • What is necessary for the activity of plasminogen is a plasminogen activator (PA).
  • PA plasminogen activator
  • uPA urokinase type
  • tPA tissue type
  • angiogenesis is an important mechanism that constitutes malignant cancer. Therefore, as with MMPs, angiogenesis in cancer tissues is considered an important target for cancer treatment strategies. In fact, it is known that the expression of vascular endothelial growth factor (VEG F) is increased in many cancers. In addition, it has been pointed out that proteases that degrade ECM have an important role not only in the above-mentioned cell migration but also in angiogenesis. Proteases, for example, create space for breaking down ECM and creating blood vessels. Thus, the malignant transformation of cancer involves intricately involved in various protease activity control systems.
  • VEG F vascular endothelial growth factor
  • protease activity factors such as PA are controlled by inhibitors.
  • uPA described above has been shown to form a complex with protein C inhibitor (PCI) in urine (Non-patent Document 1). PCI is also known to inhibit the action of uPA (Non-Patent Document 2).
  • PCI is a protease inhibitor identified as an inhibitor of protein C, an anticoagulant protease (Non-patent Document 3, Non-patent Document 4). It belongs to the serine protease (SE RPIN) family and has been confirmed to have an inhibitory action on thrombin, factor Xa, factor XIa, plasma kallikrein, and uPA. Plasminogen activator inhibitor 3 identified as an inhibitor of PA (plasminogen activator inhibitor 3) is the same molecule as PCI.
  • Non-Patent Document 1 Stump DC et al, J. Biol. Chem. 261: 12759-66, 1986
  • Non-patent document 2 Stief TW et al., Biol. Chem. Hoppe Seyler 368: 1427-33, 1987
  • Non-patent document 3 Marlar RA et al, J. Clin. Invest. 66: 1186-9, 1980
  • Non-Patent Document 4 Suzuki K. et al "J. Biol. Chem. 258: 163-8, 1983
  • An object of the present invention is to provide an anticancer agent.
  • an object of the present invention is to provide an anticancer agent having an inhibitory action on cancer cell proliferation, metastasis, and angiogenesis.
  • the present invention also relates to a method for treatment with an anticancer agent and a method for producing an anticancer agent.
  • PCI protein kinase
  • Matrigel in vitro
  • cell adhesion molecules have been reported to increase in breast cancer cells overexpressing PCI (Palmieri D. et al., J. Biol. Chem. 277: 40950-7, 2002).
  • the present inventors have examined using breast cancer cells MDA-231 cells instead of Caki-1 cells, and succeeded in observing the effect of PCI on cancer cells in vivo.
  • the present invention was completed by clarifying that PCI has the action of inhibiting the growth and metastasis of cancer cells in vivo.
  • the present inventors have found that a part of PCI's in vivo anticancer activity does not depend on PCI's protease inhibitory action, thus completing the present invention. That is, the present invention provides the following anticancer agents:
  • An anticancer agent comprising a protein C inhibitor (PCI) or a derivative thereof as an active ingredient.
  • PCI protein C inhibitor
  • the anticancer agent according to [1] which suppresses at least one selected activity of cancer growth, cancer metastasis, and angiogenesis.
  • the present invention relates to the use of a protein C inhibitor or a derivative thereof in the production of an anticancer agent.
  • the invention also relates to the use of protein C inhibitors or derivatives thereof in the treatment of cancer.
  • the present invention relates to a method for treating cancer comprising administering a protein C inhibitor or a derivative thereof. That is, the present invention provides the following:
  • a method for treating cancer comprising a step of administering a protein C inhibitor (PCI) or a derivative thereof.
  • PCI protein C inhibitor
  • the present invention provides a cancer cell growth inhibitor, a cancer metastasis inhibitor, a cancer invasion inhibitor, or an angiogenesis inhibitor in cancer tissue, comprising PCI or a derivative thereof as an active ingredient.
  • the present invention also relates to the use of PC or a derivative thereof in the manufacture of a cancer cell growth inhibitor, a cancer metastasis inhibitor, a cancer invasion inhibitor, or an angiogenesis inhibitor in cancer tissue.
  • the present invention relates to the use of PC or a derivative thereof in inhibiting cancer cell growth, inhibiting cancer metastasis, inhibiting cancer invasion, or inhibiting angiogenesis in cancer tissue. Furthermore, the present invention provides a method for inhibiting the growth of cancer cells, comprising the step of administering PC or a derivative thereof.
  • the present invention also relates to a method for inhibiting cancer metastasis, a method for inhibiting cancer invasion, or a method for inhibiting angiogenesis in cancer tissue.
  • the present invention provides an anticancer agent containing PCI or a derivative thereof as an active ingredient. According to the present invention, it has been clarified that PCI suppresses cancer invasion depending on its protease inhibitory action. Therefore, PCI derivatives having protease inhibitory action are useful as inhibitors of cancer invasion. On the other hand, the angiogenesis inhibitory effect in cancer by PCI was dependent on PCI protease inhibitory action. Similarly, the inhibition of proliferation of cancer cells by PCI was shown to be independent of the protease inhibitory action of PCI. Therefore, PCI derivatives with low or no protease inhibitory activity are useful as angiogenesis inhibitors and cancer cell growth inhibitors. It is a novel finding demonstrated by the present invention that a derivative lacking the protease inhibitory action of PCI suppresses angiogenesis and cell proliferation.
  • PCI has a strong inhibitory effect on the fibrinolytic system. Therefore, there is a concern that the administration of PCI may cause inhibition of the fibrinolytic system.
  • a PCI derivative lacking protease inhibitory action is used, a drug having no inhibitory action on the fibrinolytic system can be obtained while maintaining the anticancer action.
  • PCI inhibits activated protein C (APC) having anti-coagulant action and anti-inflammatory action, and may inhibit coagulation control system and inflammation control system in vivo.
  • APC activated protein C
  • PCI derivatives lacking protease inhibitory action are used, those concerns are eliminated, and the anticoagulant control system and inflammation control system are not affected while maintaining anticancer action, and can be used as an anticancer drug.
  • endogenous proteins having angiogenesis inhibitory action such as angiostatin and endostatin
  • endogenous angiogenesis inhibitors have a strong angiogenesis inhibitory action. Utilizing this action, development of anticancer agents targeting the angiogenic action of cancer has been underway.
  • endogenous angiogenesis inhibitors are thought to be produced by digestion of plasmin collagen by certain proteases.
  • the Oral thease inhibitors act as anticancer agents, but on the other hand have the potential to prevent the production of these angiogenesis inhibitors.
  • ADVANTAGE OF THE INVENTION According to this invention, the chemical
  • Kallistatin a heparin-binding serpin (SERPIN)
  • SERPIN heparin-binding serpin
  • anti-thrombin a hedon-binding serpin
  • AT anti-thrombin
  • angiogenesis inhibitory effect O'Reilly MS et al., Science 285: 1926-8, 1999.
  • an anticancer effect by a mechanism different from these reports, that is, a mechanism independent of the protease inhibitory action of PCI was confirmed.
  • PCI's protease inhibitor-independent anticancer effect is an action that could not be predicted from known findings.
  • FIG. 1 Photograph showing Western blot analysis of recombinant prototype PCI, R354APCI, and degPCI. 1 ⁇ g of each thread-replaceable protein (lane 1, prototype PCI; lane 2, R354APCI; lane 3, d egPCl), SDS-PAGE, and anti-human PCI rabbit IgG and then alkaline phosphatase-conjugated anti-rabbit IgG It used for the Western plot used. Each band was visualized using a Western Blue stable substrate as described in Materials and Methods.
  • FIG. 2 is a graph showing inhibition of APC by recombinant prototype PCI, R354APCI, and degPCI.
  • the inhibitory activity of each recombinant PCI against human APC was determined as follows. Each recombinant protein (40 g / ml) was incubated with human APC (40 / zg / ml) for 20 minutes in the presence of heparin (10 U / ml), and then the remaining APC was used with S-2366. Activity was determined.
  • FIG. 3A is a graph showing the effect of PCI on invasive activity of transduced! /, NA! /, MDA-231 cells.
  • Figure 3A-D shows untransfected MDA-231 cells (2 x 10 5 ), various concentrations of PCI (A), and anti-human uPA IgG (B), PAI-1 (C), or Suspended in 500 ⁇ l of DMEM in the presence of uPA (D) and placed in the culture insert (upper chamber 1).
  • the lower chamber contained DMEM 750 1 containing 10% FBS as a chemoattractant. After 24 hours of incubation, infiltrating cells on the lower surface of the membrane were fixed and stained. Cells were counted at 100X magnification under an optical microscope. Data are expressed as the number of cells derived from 4 independent infiltrating membranes (mean SD). ND indicates “not tested”.
  • FIG. 3B is a graph showing the effect of anti-human uPA IgG on the invasive activity of MDA-231 cells after transfection! /, NA! /.
  • FIG. 3C is a graph showing the effect of PAI-1 on the invasion activity of non-transfected MDA-231 cells.
  • FIG. 3D is a graph showing the effect of uPA on the invasive activity of MDA-231 cells after transformation!
  • FIG. 4 is a graph showing the effects of recombinant prototype PCI, R354APCI, and degPCI on MDA-231 cell invasion.
  • Untransfected MDA-231 cells (2 x 10 5 ) were suspended in DMEM 500 ⁇ l in the presence of 10 ⁇ g / ml prototypical PCI, R354APCI, or degPCI and the culture insert (upper chamber ).
  • the lower chamber contained DMEM 750 1 containing 10% FBS as a chemoattractant. After 24 hours of incubation, infiltrating cells on the lower surface of the membrane were fixed and stained. Cells were counted at 100X magnification under a light microscope. Data are expressed as the number of cells from 4 independent invasive membranes (mean SD). * p ⁇ 0.05.
  • FIG. 5 is a graph showing the invasion activity of prototype PCI, R354APCI, and degPCI-expressing MDA-231 cell lines.
  • MDA-PCI, MDA-R354APCI, MDA-degPCI, or MDA-Mock cell line (2 ⁇ 10 5 ) was suspended in DMEM 500 ⁇ l and placed in a culture insert (upper chamber 1).
  • the subsequent experimental procedure is basically the same as that described in the explanation of FIG.
  • Data are expressed as the number of cells derived from 4 independent infiltrating membranes (mean SD). * ⁇ ⁇ 0.05.
  • FIG. 6 is a graph showing the growth of prototype PCI, R354APCU and degPCI expressing MDA-231 cell lines in SCID mice.
  • Two ⁇ 10 5 cells of MDA-PCI, MDA-R354APCI, MDA-degPCI, or MDA-Mock in sterile DMEM 200 ⁇ l are injected intradermally into the abdominal wall of 5-week-old male SCID mice, and calipers are used. Tumor volume was measured by calipation.
  • FIG. 7 Prototype PCI, R354APCU and degPCI expressing MDA-231 cell line in SCID mice 2 is a graph and a photograph showing the experimental transition.
  • MDA-PCU MDA-R354APCU MDA-d egPCI, or MDA-Mock cell line (2 ⁇ 10 5 ) was suspended in DMEM 200 1 and injected intravenously into 5-week-old female SCI D mice. After 35 days, the lungs were removed and fixed, and the number of tumors was counted to quantify the formation of lung metastases.
  • FIG. 8 is a graph and a photograph showing a quantitative analysis of neovascularization and neovascularization in Matrigel containing prototype PCI, R354APCU and degPCI-expressing MDA-231 cell lines in SCID mice.
  • a digital camera system (Olymp us, Melville, NY) was used to photograph neovascularization in Matrigel (A).
  • FIG. 9 is a graph showing the effects of prototype PCI, R354APCI, and degPC on angiogenesis in CAM.
  • Discs containing prototype PCI, R354APCU and degPCI were mounted on embryonic serosa (CAM).
  • CAM images were taken with a digital camera system (01ympus, Melville, NY) and NIH Image 1.61 (NIH, Bethesda, MD). The new blood vessel length is measured as a pixel, and the result is expressed as a mean value S.D. * p ⁇ 0.05.
  • FIG. 10 is a graph and a photograph showing the effects of prototype PCI, R354APCU and degPCI on HUVEC tube formation.
  • HUVEC (2 x 10 4 cells) in MCDB-131 supplemented with 5% FBS on a 24well plate coated with Matrigel in the presence of 10 g of prototype PCI, R354APCI or degPCI, or These were cultured in the absence of PCI (control).
  • Photographs were taken 6 hours after plating (magnification X 100) (A) and quantitative analysis of the effects of various recombinant mutant PCIs on tube formation (B). The tube length is measured in pixels, and the result is expressed as an averager SD. * p ⁇ 0.05.
  • PCI exhibits an anticancer effect in vivo.
  • the anticancer effect is brought about by suppression of cancer cell proliferation, invasion, and angiogenesis.
  • the existence of multiple PCI derivatives that maintained these anticancer effects was revealed. Based on these findings, an anticancer agent containing PCI or a derivative thereof as an active ingredient has been provided.
  • PCI is a single-chain glycoprotein with a molecular weight of about 57 kDa.
  • natural PCI and artificially produced PCI can be used.
  • PCI of humans and other mammals is known (Suzuki, K. et al "J. Biol. Chem. 262: 611-6, 1987)
  • human-derived PCI is The amino acid sequence of human PCI is shown in SEQ ID NO: 2 (GenBank Accession Number: P05154), and the nucleotide sequence of cDNA is shown in SEQ ID NO: 1.
  • PCI derived from a species other than human is used.
  • PCI such as mouse, rat or ushi
  • PCI derivative a polypeptide containing a PCI fragment sequence that maintains anticancer activity.
  • the polypeptide containing the full length of PCI, or the polypeptide containing the fragment sequence thereof includes a fusion polypeptide modified with other peptides.
  • PCIs may be used as a term that includes derivatives that maintain their anticancer activity.
  • a PCI fragment having low or no protease inhibitory activity can be used as an active ingredient of an anticancer agent for suppressing cancer metastasis and / or growth.
  • low protease inhibitory activity refers to protease inhibitory activity of, for example, 50% or less, or 30% or less, usually 10% or less, compared to natural PCI.
  • the inhibitory activity of a PCI derivative against a protease can be evaluated by, for example, a method as shown in the Examples. That is, activated protein C (A PC) is incubated with a PCI derivative whose inhibitory effect is to be evaluated. By comparing the protease activity after incubation, the protease inhibitory activity of PCI or its derivative can be evaluated. Protease activity can be measured by giving an appropriate substrate and using the amount of substrate digested by the protease or the rate of digestion as an indicator. Digestion can be traced optically using a chromogenic substrate with a dye attached to the peptide. In the examples, Glu-Pro-Arg-p-nitroanilide (S-2366) was used as the chromogenic substrate.
  • aPC activated protein C
  • aPC synthesized by genetic engineering or chemically can be used.
  • aPC not only the full-length amino acid sequence but also a fragment maintaining the enzyme activity can be used.
  • the amino acid sequences of human and other mammalian aPCs are known (Mather, T. et al., EMBO J. 15: 6822-6 831, 1996; Foster, DC et al., Proc. Natl. Acad. Sci. 82: 4673-4677, 1985).
  • commercially available aPC protein C activated, from human plasma, SIGMA, # P2200 can also be used.
  • PCI derivative having low protease inhibitory activity for example, the following polypeptides can be shown.
  • a polypeptide having these conditions is useful as an active ingredient of an anticancer agent in the present invention.
  • PCI derivatives useful in the present invention will be specifically described.
  • a PCI mutant lacking the protease inhibitory action in the present invention for example, a polypeptide containing a binding region of PCI with heparin or heparin-like glycosaminodalcan can be shown.
  • the following regions are considered to be important for binding to heparin (Kuhn LA et al, Proc Natl Acad Sci USA. 87:85 06-8510, 1990).
  • both amino acid sequences can be aligned to identify a region homologous to the region.
  • a PCI fragment containing this region can be used as a PCI derivative in the present invention.
  • a fragment amino acid sequence for example, a fragment containing the amino acid residue at position 1354 on the N-terminal side is shown. You can.
  • the fragment (SEQ ID NO: 15) described as degPCI in the Examples is an amino acid sequence corresponding to position 1354 of SEQ ID NO: 2, and has sufficient anticancer activity while having no protease inhibitory activity. It was confirmed.
  • Table 1 also shows the distribution of these heparin-like dalcosaminodalcans in vivo. PCI derivatives that bind to these dalcosaminodalcans are useful in the present invention.
  • chondroitin sulfate has a large effect of enhancing the action of PCI.
  • dextran sulfate is known to enhance the action of PCI as well as heparin (Kazama Y. et al, Thromb Res. 48 (2): 179-85, 1987 Oct 15). Therefore, a variant of PCI that maintains binding to chondroitin sulfate and dextran sulfate is preferred in the present invention.
  • the binding property of PCI derivative with henolin or henolin-like glycosaminodalcan can be evaluated by any method.
  • henones are used as terms to indicate heparin and henoline-like glycosaminoglycan.
  • the binding of PCI derivatives to heparins can be assessed by observing the binding of PCI derivatives to immobilized heparins.
  • PCI derivatives for heparin using heparin columns Can be compared. In other words, PCI or PCI derivatives are adsorbed on a heparingarose column and eluted with NaCl.
  • PCI or PCI derivatives contained in the eluted fraction can be quantified by E LISA or the like, and the adsorption level on the column can be compared.
  • PCs and PCI derivatives with high adsorption activity to heparin are eluted in a slower fraction (Kuhn LA et al, Proc Natl Acad Sci USA. 87: 8506-8510, 1990).
  • PCI derivative in the present invention a mutant PCI having a mutation at the digestion site by aPC can also be used.
  • PCI binds to aPC and inactivates
  • PCI is digested by aPC in the region close to its C-terminal, and the C-terminal fragment is released.
  • the part cleaved by aPC has been shown to be between Arg-Ser at positions 354-355 (Suzuki K. et al., J. Biol. Chem. 262 (2): 611-616, 1987).
  • PCI derivatives containing mutations in this region are not digested by aPC and lose their protease inhibitory activity.
  • the PCI mutant R354APCI shown in the Examples is a PCI derivative in which Arg at position 354 is mutated to Ala.
  • R354 4APCI does not have protease activity because it contains a mutation at the site of extinction by aPC.
  • Such a mutant can also be used as a PCI derivative in the present invention.
  • PCI in the present invention may be a polypeptide derived from various species other than human in addition to the human polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.
  • the PCI derivative in the present invention may be a polypeptide containing an amino acid sequence in which one or more amino acid residues are modified in the amino acid sequence of PCI as long as it has an anticancer activity.
  • a modification of the amino acid sequence may be a deletion, substitution or addition of one or more amino acids in the amino acid sequence.
  • the amino acid sequence can be modified to improve in vivo stability or the physical and biological properties of PCI. Methods for modifying amino acid sequences are known.
  • polypeptides whose amino acid sequences have been modified by methods such as site-specific mutation (see Kunkel TA, Proc. Natl. Acad. Sci. USA 82: 488-92, 1985), PCR mutation, cassette mutation, etc. Can get the DNA that encodes it.
  • Such a variant has at least 70%, more preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, even more preferably with the original amino acid sequence.
  • sequence identity refers to residues that are the same as the residues of the original PCI amino acid sequence after aligning the sequences as necessary to maximize sequence identity and introducing gaps as appropriate. Is defined as the percentage of
  • amino acid residue When the amino acid residue is modified, the property of the protein can be maintained by substituting it with another amino acid residue in which the property of the amino acid side chain is conserved.
  • amino acids having the following characteristics have similar characteristics to each other, and the protein activity is likely to be maintained when they are substituted with each other.
  • the alphabets in parentheses all represent one letter of amino acids.
  • Amino acids with aliphatic side chains (G, A, V, L, I, P)
  • Amino acids with side chains containing carboxylic acids and amides (D, N, E, Q)
  • substitutions of amino acids within each of these groups are referred to as conservative substitutions.
  • a polypeptide having an amino acid sequence modified by deletion, addition and substitution of one or more amino acid residues to a certain amino acid sequence and substitution by Z or other amino acids retains its biological activity.
  • already known Mark, DF et al., Proc. Natl. Acad. Sci. USA 81: 5 662-6, 1984; Zoller, MJ and Smith, M., Nucleic Acids Res. 10: 6487-500, Wang, A. et al "Science 224: 1431-3, 1984; Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA 79: 6409-13, 1982).
  • the number of amino acids to be mutated is not particularly limited, and is usually within 40%, preferably within 35%, more preferably within 30% (for example, within 25%) of the amino acids constituting the full-length amino acid sequence.
  • the identity of amino acid sequences can be determined by the following method.
  • PCI and a derivative maintaining its anticancer activity can be used.
  • the anticancer activity of PCI and PCI derivatives can be confirmed, for example, by the method as shown in the Examples. Specifically, the following methods can be used to confirm the effects on cancer cell invasion, proliferation, metastasis, or angiogenesis. Using these methods, the anticancer activity of the candidate polypeptide is confirmed, and a polypeptide that has confirmed at least one anticancer activity of invasion, proliferation, metastasis, or angiogenesis is selected as a PCI derivative in the present invention. can do. Anticancer activity can be evaluated by comparison with a reference polypeptide that does not have anticancer activity.
  • the vector (Mock) without the DNA can also be used as a control.
  • the vector (Mock) without the DNA can also be used as a control.
  • a polypeptide that has been confirmed to have anticancer activity it can be confirmed by confirming that it has the same or higher anticancer activity.
  • Methodastasis assay of experimental lung metastasis
  • the in vivo evaluation system can be used to evaluate the effects of PCI derivatives on cancer cell metastasis.
  • breast cancer cells MDA-231 can be inoculated into the tail vein of SCID mice and observed for metastasis to the lung. If the metastasis to the lung is suppressed by transformation of DNA encoding the PCI derivative, the inhibitory effect on the metastasis of the PCI derivative can be confirmed.
  • the effect of PCI derivatives on angiogenesis in cancer tissues can also be evaluated in vivo. That is, Matrigel containing cancer cells can be transplanted into SCID mice, and the angiogenic activity of cancer cells can be evaluated using the entry of blood components into Matrigel as an index. In the examples, Matrigel was digested and the content of hemoglobin contained in Matrigel was compared. If angiogenesis is inhibited in cancer cells transformed with DNA encoding a PCI derivative, the inhibitory effect of the PCI derivative on angiogenesis can be confirmed.
  • the angiogenic activity of the cancer cells can be evaluated. If angiogenesis is suppressed in cancer cells transformed with DNA encoding a PCI derivative, the inhibitory action of the PCI derivative on angiogenesis can be confirmed.
  • the angiogenic activity of cancer cells can be evaluated using the capillaries formed by the cancer cells in Matrigel as an index (Schnaper HW. Et al., J. Cell Physiol 65: 107-18, 1995). If angiogenesis is suppressed in cancer cells transformed with DNA encoding a PCI derivative, the PCI derivative can The inhibitory action can be confirmed.
  • the present invention by using breast cancer cell MDA-231 as a cancer cell, activities such as proliferation, invasion, metastasis, and angiogenesis of the cancer cell can be evaluated in vivo. That is, by transplanting MDA-231 into SCID mice, the malignant mechanisms of cancer such as proliferation, invasion, metastasis, and angiogenesis can be experimentally reproduced. Therefore, the present invention provides a method for evaluating the anticancer activity of a test substance, which comprises the following steps.
  • the activity as a cancer cell can be selected as a group force including cell proliferation, invasion, metastasis, and angiogenic potential. These activities can be measured, for example, by the method as described above.
  • MDA-231 cells used in the above method can be obtained from the cell bank as MDA-MB-231 (ATCC Accession No. HT B-26).
  • PCI can be isolated from nature using blood, urine, seminal plasma, synovial fluid, or cells or tissues that express PCI as a raw material based on its physical properties. Alternatively, it may be chemically synthesized based on known sequence information.
  • PCI derivatives can be synthesized by genetic engineering by expressing DNA encoding the target amino acid sequence. That is, host cells can be transformed with a gene encoding PCI or a PCI derivative by gene recombination technology. The gene is preferably incorporated into an appropriate expression vector and transformed into a host cell. By culturing the obtained transformed cells, PC or PCI derivatives can be obtained from the cells or the culture supernatant thereof. A recombinant of PCI or a PCI derivative can be prepared, for example, as described in the Examples.
  • a vector suitable for producing a PC derivative or PCI derivative by genetic engineering Various vectors using Rus, cosmids, plasmids, and nocteriophages can be used (Molecular Cloning 2 ed., Cold Spring Harbor Press (1989); Current Pro tocols in Molecular Biology, John Wiley & Sons ( 1987)) 0 to PCI is expressed when introduced into a desired host cell, the vector may contain appropriate control sequences.
  • a gene encoding PCI or a PCI derivative is inserted into a vector so that the reading frame does not deviate from the control sequence.
  • the gene or base sequence of the gene encoding the PC or PCI derivative is arbitrary as long as it can be expressed in the selected vector and host.
  • RNA can also be used in some cases.
  • control sequence suitable for expression can be appropriately selected depending on the host cell or vector.
  • the vector contains at least a promoter, a ribosome binding site and a terminator as control sequences.
  • the essential regulatory sequence is a promoter and a terminator.
  • an enhancer, a splicing signal, a transcription factor, a transactivator, a poly A signal and / or a polyadenylation signal can be included as necessary.
  • a vector for expressing a PC or PCI derivative may further contain a selectable marker, if necessary. Host cells transformed with the marker can be easily selected.
  • the signal peptide coding sequence can be transferred to the PCI gene or PCI derivative to transduce intracellularly expressed PCI or PCI derivatives into the endoplasmic reticulum lumen or extracellular, or into periplasm if the host is a Gram-negative bacterium. It may be incorporated into a vector so as to be added to a PCI derivative gene.
  • Such a signal peptide may be unique to PCI, as long as it is recognized correctly in the selected host cell, or may be derived from a heterologous protein.
  • a linker a start codon, a stop codon, etc. may be added.
  • the N-terminal position 119 is a signal sequence unique to human PCI. Therefore, the amino acid sequence in this region can be recombined with other signal sequences.
  • the gene may be inserted into the vector by a ligase reaction using a restriction enzyme site.
  • Can force S Molecular Cloning 2 n ed, Cold Spring Harbor Press (1989) Section5.61- 5. 63;. Current Protocols in Molecular Biology, John Wiley & Sons (1987) 11.4-11.11.
  • a vector can be designed by selecting a base sequence that provides high expression efficiency (Grantham R. et al., Nucleic Acids Res. 9: r43). -74, 1981).
  • PCI or a PCI derivative is produced by introduction into an appropriate host
  • a combination of the above expression vector and an appropriate host can be used.
  • eukaryotic cells animal cells, plant cells, and fungal cells can be used. Examples of cells that can be used as hosts are shown below.
  • Mammalian cells For example, CHO, COS, myeloma, BHK (baby hamster kidney), He La, Vero,
  • Amphibian cells Xenopus oocytes, for example
  • Insect cells For example, S19, S121, Tn5, etc., or individuals such as silkworms
  • Nicotiana genus for example, calli culture of cells from Nicotiana tabacum
  • Yeast for example, Saccharomyces genus; eg Saccharomyces serevisiae
  • Filamentous fungi for example, Aspergillus genus; for example Aspergillus niger
  • Bacterial cells for example, Escherichia coli, Bacillus subtilis
  • PC or PCI derivatives can be obtained by introducing the PCI gene or PCI derivative gene into these cells by transformation and culturing the transformed cells in vitro.
  • a method suitable for the selected host and vector may be employed.
  • calcium treatment and electroporation The method by the method etc. is known.
  • the agrobacterium method can be used, and for mammalian cells, the calcium phosphate precipitation method can be used.
  • the present invention is not particularly limited to these methods, but known nuclear microinjection, cell fusion, electric pulse perforation, protoplast fusion, lipophectamine method (GIBCO BRL), DEAE-dextran method, FuGENE6 reagent (Boehringer- Various methods including the method using Mannheim) can be adopted.
  • Host cell culture can be performed according to a known method suitable for the selected cell.
  • a medium such as DMEM, MEM, RPMI_1640, 199 or IMD M
  • FCS urine fetal serum
  • necessary operations such as medium exchange, aeration, and agitation can be performed as needed during culture.
  • PCI and PCI derivatives are preferably purified and used by a known method.
  • PCI or PCI derivatives can be purified uniformly according to general protein purification methods. For example, the following purification techniques can be selected as appropriate and combined to separate and produce the target protein with S (Strategies for Protein Purification and Charcterization: A Laboratoy Course Manual, Daniel R. Marshak et al. Eds , Cold Spring Harbor Laboratory Press (1996); Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)).
  • the purification method in the present invention is not limited to these. Further, as a chromatograph that can be used in the present invention, the following chromatography can be shown.
  • chromatographies can be performed using liquid phase chromatography such as HPLC and FPLC.
  • an antibody against PCI can be used in the affinity chromatography.
  • the anticancer agent of the present invention contains a PC or PCI derivative obtained as described above as an active ingredient.
  • a single PCI or PCI derivative may be blended as an active ingredient, or multiple types of PCI and Z or PCI derivatives may be blended.
  • “Containing PCI and Z or a PCI derivative as an active ingredient” means containing PCI and Z or a PCI derivative as at least one active ingredient.
  • the content of the active ingredient in the anticancer agent is not limited.
  • the anticancer agent of the present invention can contain other active ingredients having anticancer activity in combination with PCI and Z or PCI derivatives.
  • PCI and PCI derivatives can be formulated according to a conventional method (for example, Remington Pharmaceutical Science, latest edition, Mark Publishing company, Easton, U.3 ⁇ 4. A).
  • a pharmaceutically acceptable carrier and / or additive can be included as required.
  • surfactants PEG, Tween, etc.
  • excipients antioxidants (ascorbic acid, etc.)
  • coloring agents flavoring agents, preservatives, stabilizers, buffering agents (phosphoric acid, citrate, other organics) Acid), chelating agents (EDTA, etc.), suspending agents, tonicity agents, binders, disintegrating agents, lubricants, fluidity promoters, flavoring agents, and the like.
  • the anticancer agent of the present invention can also contain other conventional carriers.
  • PCI and Z or PCI derivatives are dissolved in an isotonic solution containing, for example, physiological saline, glucose or other adjuvants.
  • Adjuvants include, for example, D-sorbitol, D-mannose, D-mannitol, sodium chloride, and further suitable solubilizers such as alcohol (ethanol, etc.), polyalcohol (propylene alcohol, PEG, etc.), nonionic surfactants (polysorbate 80, HCO-50), etc. may be used in combination.
  • PCI and Z or PCI derivatives can be encapsulated in microcapsules (microcapsules such as hydroxymethylcellulose, gelatin, poly [methylmethacrylic acid]) or colloid drug delivery systems (ribosomes, albumin micross). Fuea, Maikuroe Marujiyon may be a nano-particles, and nano-capsules) ( "Remington's Pharmace utical Science 16 th edition", Oslo Ed. (1980) see the like). Furthermore, a method of making a drug a sustained-release drug is also known (Langer et al., J. Biomed. Mater. Res. 15: 167-277, 1981; Langer, Chem. Tech. 12: 98-105).
  • the anticancer agent of the present invention can be administered by either oral or parenteral routes.
  • a preferred method of administration is parenteral administration. Specifically, it is administered to patients by injection, nasal administration, pulmonary administration, and transdermal administration.
  • Examples of the injection form can be administered systemically or locally by, for example, intravenous injection, intramuscular injection, intraperitoneal injection or subcutaneous injection.
  • the administration method can be appropriately selected depending on the age and symptoms of the patient.
  • the dose can be selected, for example, in the range of 0.0001 mg to 1000 mg per kg body weight. Alternatively, for example, the dose can be selected in the range of 0.001 to 100,000 mg / body per patient.
  • the dose of the anti-cancer of the present invention is not limited to these doses.
  • a gene encoding the above-described PCI and Z or PCI derivative can be incorporated into a vector therapy vector to provide an anticancer drug for gene therapy.
  • a gene administration method in addition to direct administration using a naked plasmid, it can be packaged in a ribosome or the like.
  • various gene therapy vectors can be used. For example, the following various viral vectors for gene therapy are known (see Adolph KW ed., Viral Genome Methods, CRC Press, Florid (1996)).
  • the gene can be coated on a bead carrier such as colloidal gold particles (WO93 / 17706 or the like).
  • the gene administration method is not limited as long as PCI or a PCI derivative is expressed in vivo and can exert its action.
  • a sufficient amount is administered by injection or infusion of the gene via a suitable parenteral route.
  • Parenteral routes include intravenous, intraperitoneal, subcutaneous, intradermal, adipose tissue, mammary tissue, inhalation or intramuscular route, gas-induced particle bombardment method (using an electron gun, etc.), nasal medication, etc. The method through the mucosal route can be shown.
  • a gene encoding a PC or PCI derivative can also be administered by introducing a gene into a cell ex vivo and returning the cell to an animal.
  • ribosome transformation for ex vivo gene introduction, ribosome transformation, particle bombardment (US Pat. No. 4,945,050), viral infection, or the like can be used.
  • Restriction endonuclease was purchased from TOYOBO, Japan, Osaka. Taq DNA Polymer The cases were from Roche Biochemicals (Basel, Switzerland), and the Klenow fragment, T4 polynucleotide kinase, and T4 DNA ligase were from Nippon Gene (Tokyo, Japan).
  • QuikChange XL site-directed mutagenesis kit was purchased from Stratagene, Cedar Creek, Texas, USA.
  • the Die Terminator One-Cycle Sequence Lady Reaction Kit was purchased by ABI, Foster City, California. Radionucleotide ([a - 32 P] dCTP and [ ⁇ - 32 ⁇ ] ⁇ ) were from Amersham Bioscience (Sweden, Upusa la).
  • Transfusion reagent Effectene was purchased from QIAGEN, Inc., Tokyo, Japan. All other chemicals and reagents used were of the highest quality on the market o
  • MDA-231 was obtained from Japan Cancer Research Resource Bank. MDA-231 is Dulbecco supplemented with 10% urine fetal serum (FBSKEQUITECH-BIO, Kelville, TX), 100 ⁇ g / ml penicillin, and 100 IU / ml streptomycin (Sanko Junyaku, Tokyo, Japan). Modified Eagle Medium (DMEM) (Nissui Pharmaceutical, Tokyo, Japan) with 5% CO
  • R354APCI cDNA with a signal peptide is a full-length PCI cDNA (Suzuki K. et al., J. Biol. Chem. 262: 611-6, 1987) using a synthetic oligonucleotide primer consisting of the following nucleotide sequences: As prepared with the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, Calif.). Underlined nucleotides indicate mutation sites.
  • a degPCI cDNA having a signal peptide sequence was prepared by polymerase chain reaction (PCRXSaiki RK et al., Science 239: 487-91, 1988) using the following primer pairs.
  • the primers represented by SEQ ID NO: 5 and SEQ ID NO: 6 correspond to positions 31 to 52 and positions 1194 to 1176 of PCI cDNA (SEQ ID NO: 1), respectively.
  • Underlined nucleotides indicate the EcoRI site inserted at the 5 'end of the two primers.
  • mutant R354APCI cDNA, or degPCI cDNA was transformed into the mammalian expression vector pRC / CMV (Invitrogen Corp. , Carlsbad, Calif.) And transfected into MDA-231 using Effectance transfection reagent.
  • a prototype PCI, R354APCU degPCI expressing MDA-231 cell line was selected using DMEM containing 800 ⁇ g / ml dieticin. Then, as shown below, the PCI antigen in the medium of each cell line was measured by enzyme immunoassay (ELISA) and by PCI mRNA evaluation by Northern blot analysis, and the prototype PCI of the cloned MDA-231 cell line , R354APCI, or degPCI expression was confirmed.
  • ELISA enzyme immunoassay
  • MDA-PCI 1 and MDA-PCI 2 as cell lines expressing large amounts of PCI
  • MDA-R354APCI 1 and MDA-R354APCI 2 as cell lines expressing large amounts of R354APCI
  • MDA-degPCI 1 and MDA-degPC I 2 were selected as cell lines expressing a large amount of degPCI and used in the experiment.
  • MDA231 cells transfected with pRC / CMV without DNA insert were prepared and used as negative controls and were designated MDA-Mock 1 and MDA-Mock 2.
  • PCI and uPA antigen in the medium were determined as described previously (Wakita T. et al., Int. J. Cancer 108: 516-23, 2004), using enzymes with polyclonal anti-PCI and anti-uPA antibodies. Determined by immunoassay (ELISA).
  • RNAzol B reagent TEL-TEST, Friendswood, TX
  • Total RNA was prepared from MDA-231, MDA-PCU, MDA-R354 APCI, MDA-degPCI, and MDA-Mock cells using the acid guanidine-phenol closed mouth technique (Chomczynski P. et al., Anal Biochem 162 : 156-59, 1987).
  • Total RNA was quantified spectrophotometrically and stored at -80 ° C until use.
  • RNA (20 ⁇ g) from MDA-231, MDA-PCI, MDA-R354APCI, MDA-degPCI, and MDA-Mock cells was electrophoresed in formaldehyde-agarose gel, and then GeneScreen nylon membrane (NEN Life Science, Boston, Massachusetts). After UV crosslinking, the membrane was hybridized with a random prime 32 P-labeled full-length human PCI cDNA probe (Suzuki K. et al., J. Biol. Chem. 262: 611-6, 1987).
  • the membrane was boiled in 0.5% SDS and the 32 P-labeled human glyceraldehyde triphosphate dehydrogenase (GAPDH) cDNA probe (Clontech, Power Refolder) as described above. Palo Alto).
  • GPDH human glyceraldehyde triphosphate dehydrogenase
  • RT-PCR was performed to evaluate the expression of a small amount of human PCI in MDA-231 cells.
  • Total RNA (5 ⁇ g) from which MDA231 cell force was also extracted was reverse transcribed using oligo dT primer and Superscript first strand cDNA synthesis kit (Invitrogen Corp., Carlsbad, Calif.) According to the manufacturer's instructions.
  • the nucleotide sequences of the forward and reverse PCR primers used for human PCI amplification are as follows.
  • the primers shown in SEQ ID NO: 7 and SEQ ID NO: 8 correspond to positions 733 to 752 and positions 1289 to 1270 of PCI cDNA (SEQ ID NO: 1), respectively (Suzuki K., J Biol. Chem. 262: 611-6, 1987)
  • the PCR mixture was composed of PCR buffer (100 mM Tris-HC1, 200 mM KC1, pH 8.3), 25 mM Mg CI, 200 ⁇ M deoxyribonucleoside (dATP, dGTP, dCTP, dTTP), respectively. 0.2 ⁇
  • Wild-type PCI cDNA containing no signal peptide sequence was prepared by polymerase chain reaction (PCR) using the following primer pair (Saiki R.K. et al., Science 239: 487-91, 1988).
  • the primers shown in SEQ ID NO: 9 and SEQ ID NO: 1 correspond to positions 133-152 and 1296-1276 of PCI cDNA (SEQ ID NO: 1), respectively.
  • Underlined nucleotides indicate the inserted BamHI site (Hayashi T. et al., Int. J. Hemato 1. 58: 213-224, 1993).
  • DegPCI cDNA also uses the following primer pair: Prepared by PCR.
  • the primers shown in SEQ ID NO: 11 and SEQ ID NO: 12 correspond to positions 133 to 152 and 1194 to 1175 of the PCI cDNA (SEQ ID NO: 1), respectively.
  • Underlined nucleotides indicate the inserted BamHI site (Hayashi T. et al "Int. J. Hematol. 58: 213-224, 1993).
  • the activity of various recombinant PCIs was determined as follows. APC (40 g / ml) 101 was mixed with TBS 10 ⁇ l containing 30 U / ml heparin and recombinant prototype PCI, R354APCI, and degPCI (40 ⁇ g / ml O / z 1. The mixture was 37 After 20 minutes of incubation at ° C, it was mixed with 250 ⁇ l of buffer containing 0.2 mM S-2366, 50 mM Tris-HCl (pH 8.0), and 0.1 M CsCl After the mixture was incubated at room temperature for 15 minutes The reaction was stopped by adding 100% acetic acid 251. Absorbance was measured at 405 nm using a model 550 microplate reader (Bio-RAD) A calibration curve was prepared using serial dilutions of APC. Residual APC activity was calculated.
  • each insert membrane was gently rubbed with a cotton swab to remove any non-soaked cells and Matrigel. Infiltrating cells on the lower surface of the membrane were fixed and stained using the Diff-Quik staining kit (International Reagent, Kobe, Japan). Cells were counted at 100X magnification under a light microscope.
  • mice Male and female severe combined immunodeficient mice (SCID) (5 weeks old) were purchased from CLEA Japan (Osaka, Japan). Mice were housed in a constant cycle of 12 hours light / 12 hours dark, allowing free access to standard food and water. The experiment was approved by the Mie University Animal Experiments Review Committee and conducted in accordance with the laboratory animal guidelines of the National Institutes of Health.
  • Subconfluent E cement of MDA- PCI, MDA-R354APCU MDA- degPCI , or MDA Mo ck cells were harvested with EDTA solution, the appropriate density (2.5 x 10 6 cells / ml) with serum-free DMEM re Suspended.
  • MDA-PCU MDA-R354APCI, MDA-degPCI, or MDA-Mock cells were then injected into the tail vein of female SCID mice. Thirty-five days after tumor injection, the mice were anesthetized with pentobarbital and sacrificed. Lungs were removed and fixed in formaldehyde neutral buffer. Using a magnifying glass, a small blob that appeared as white spots was counted.
  • MDA-PCI MDA-R354APCI
  • MDA-degPCI MDA-degPCI
  • MDA-Mock cells 2 x 10 6 cells / ml
  • VEGF vascular endothelial growth factor
  • 0.5 ml of Matrigel 9-10 mg / ml; Becton Diskinson, Franklin Lake, NJ
  • centripetal capillaries on Matrigel plugs were visualized using a digital camera system (Olympus, Melville, NY).
  • the Matrigel plug was digested with 1 ml of 0.1% collagenase dissolved in Hank's solution, and a new blood vessel was obtained using the hemoglobin atsey method (Passaniti A. et al., Lab. Invest. 67: 519-28, 1992). The number of was quantified.
  • a square plastic wrap was fixed to an open plastic tube with a ring (diameter 8 cm, rear 6 cm) to prepare an egg hammock.
  • the fertilized egg (3 days old) was divided, the contents were transferred to an egg hammock, and the embryo was manipulated so that it was on the surface of the egg.
  • a Petri dish lid was placed over the hammock.
  • Embryos were maintained in a 37 ° C humidified incubator.
  • 2% methylcellulose 50 ⁇ l was dried on a Petri dish to prepare a methylcellulose disk.
  • the disc was peeled off from the pan and placed on the CAM, and then the prototype PCI, R354APCU or degPCI was added onto the disc.
  • CAM images were taken with a digital camera system (Olympus, Melville, NY) and NIH Image 1.61 (NIH, Bethesda, MD). Of 100x random, unaffected blood vessels in 6 fields of view Counted the number.
  • Example 1 Expression, purification, and APC inhibitory activity of recombinant prototype PCI, R354APCI, and degPCI produced by baculovirus expression system
  • Prototype PCI, R354APCI and degPCI expressed by baculovirus expression system were purified using Hi trap CM FF and Hitrap heparin FF. The purified protein was then detected by Western blot analysis under reducing conditions. The MW of the prototype PCI, R354APCI, and degPCI were approximately 57 KDa, approximately 57 KDa, and approximately 54 KDa, respectively ( Figure 1). These recombinant PCIs were also tested for APC inhibitory activity. R354APCI and degPCI were almost unable to inhibit APC ( Figure 2).
  • MDA-231 cells Effect of prototype PCI on the invasiveness of MDA-231 cells in the Matrigel system.
  • uPA and PCI expression in MDA-231 cells was evaluated by ELISA and RT-PCR analysis.
  • MDA-231 cells are 470 ng / 10 4 cells / 24 h uPA, respectively, and 58 ng / 10 4 Cells / 24 hours of PCI expression.
  • RT-PCR analysis also indicated that both uPA mRNA and PCI mRNA were expressed by MDA-231 cells (data not shown). Subsequently, the effect of PCI on the invasive activity of MDA-231 cells was tested.
  • Prototype PCI significantly inhibited the invasion activity of MDA-231 cells in a dose-dependent manner (Figure 3A), but BSA was potent in effect on this activity (data not shown). Furthermore, anti-human PCI antibodies blocked PCI-induced inhibition of MDA-231 cell invasion in a dose-dependent manner (data not shown). Invasion assay also showed that the invasiveness of MDA-231 cells was inhibited by anti-uPA antibodies ( Figure 3B) and PAI-1 ( Figure 3C) and promoted by uPA in a dose-dependent manner ( Figure 3D).
  • MDA-PCI, MDA-R354APCI, and MDA-degPCI cell lines showed strong expression of each PCI mRNA, whereas untransfected MDA and MDA-Mock cell lines expressed weakly the prototype PCI. / !, (data not shown).
  • the amount of PCI secreted by MDA-P CI 1 and MDA-PCI 2 cells was 10.3 and 12.5 ng / 10 4 cells / 24 hours, respectively.
  • MDA-R354APCI 1 and MDA-R354APCI 2 cells The amount of R354APCI secreted was 20.0 and 12.0 ng / 10 4 cells / 24 hours, respectively.
  • the amounts of degraded PCI secreted from MDA-degPCI 1 and MDA-degPCI 2 cells were 3.0 and 1.1 ng / 10 4 cells / 24 hours, respectively.
  • the growth rate and uPA production of these cell lines were almost the same (data not shown).
  • FIG. 8A shows that angiogenesis on Matrigel containing PCI-expressing MDA-231 cells and R354APCI and degPCI-expressing MDA-231 cells was lower than Mock-expressing MDA-23 1 cells.
  • FIG. 8B hemoglobin levels in Matrigel containing MDA-PCI cells, MDA-R354 APCI or MDA-degPCI cell lines were significantly lower than those of MDA-Mock cell lines.
  • PCI significantly inhibited vascular growth in the ⁇ -bird CAM assembly.
  • PCI-treated CAMs were significantly smaller than controls and had less angiogenesis.
  • R354APCI and degPCI also significantly inhibited vascular growth.
  • PCI expression is significantly reduced in renal cancer cells compared to non-neoplastic renal tissue, and that PCI expression is infiltrating the renal cancer cell line Caki-1 cells. It has been demonstrated to inhibit activity (Wakita T. et al., Int. J. Cancer 108: 516-23, 2004).
  • PCI-expressing Caki-1 cells were used to assess the effect of PCI on tumor growth and metastasis in vivo. Even wild-type Caki-1 cells were unable to grow in SCID mice. Based on this, the effect of PCI on the invasiveness of breast cancer cell line MDA-231 cells was tested. MDA-23 1 cells expressed large amounts of uPA and small amounts of PCI!
  • PCI inhibitory activity was weaker than that of Caki cells. This result is probably due to uP expression A by MDA-231, which is much higher than the Caki-1 cells. Furthermore, the invasive activity of MDA-231 cells was increased by uPA supplementation and inhibited by PAI-1. This result is consistent with the notion that MDA-23 1 infiltration is primarily mediated by uPA.
  • the present inventors also evaluated the effect of PCI on the invasive activity of B 16 mouse melanoma cells and found similar results (data not shown). These findings suggest that the invasion of various tumor cells is controlled by PCI.
  • PCI-expressing MDA-231 cells are less invasive than Mock transfection MDA-23 1 cells. This finding is in agreement with previous data showing that PCI-expressing Caki-1 cells are less invasive than Mock Transfection Cali-1 cells.
  • the present inventors also described PCI protease inhibitory activity on the invasion ability of MDA-231 cells. The invasive ability of degPCI-expressing MDA-231 cells and R354APCI-expressing MDA-231 cells was not significantly different from that of Mock-expressing MDA-231 cells. This result is a recombination 13 ⁇ 454? This is consistent with the data showing that 16 ⁇ 1 is ⁇ 1 "0 and has no effect on the invasion of ⁇ 10 -231 cells.
  • Kallistatin is a heparin-binding serpin that inhibits tumor growth through suppression of angiogenesis (Miao R.Q. et al., Blood 100: 3245-52, 2002).
  • VEGF and bFG F are potent mediators of angiogenesis, and the binding power of VEGF and bFGF to heparan sulfate proteoglycans on the surface of endothelial cells regulates the binding of VEGF and bFGF to specific receptors are well known (Folkman J. et al., Adv. Exp. Med. Biol. 31 3: 355-64, 1992). Heparin itself has also been reported to promote SVEGF-dependent angiogenesis (Folkman J. et al., Adv. Exp. Med. Biol. 313: 355-64, 1992).
  • Calistatin has the ability to inhibit angiogenesis through its ability to bind henoline. This binding is important for VEGF to bind to its receptor, and therefore, it inhibits VEGF-mediated angiogenesis ( Miao RQ et al., Am. J. Physiol. Cell Physiol. 284: C16 04-13, 2003).
  • a latent form of antithrombin (AT), a heparin-binding serpin, is also known to have anti-angiogenic activity (O'Reilly MS et al., Science 285: 1926-8, 1999). It has recently been reported that AT down-regulates the expression of proangiogenetic proteolycan, perlecan, in endothelial cells (Zhang W.
  • PCI is also a heparin-binding serbin.
  • the inventors have found that PCI inhibits MDA-231 proliferation and metastasis and thus evaluated the effect of PCI on angiogenesis.
  • the present invention demonstrates that PCI inhibits tumor growth and metastasis in vivo, and that the anti-metastatic and anti-proliferative activity of PCI is independent of its protease inhibitory activity. did. These findings suggest that PCI is potentially useful for the treatment of various tumors such as renal cell carcinoma and breast cancer.
  • PCI suppresses cancer invasion depending on its protease inhibitory action. Therefore, PCI derivatives having protease inhibitory action are useful as inhibitors of cancer invasion.
  • angiogenesis inhibitory effect in cancer by PCI was dependent on PCI protease inhibitory action.
  • the inhibition of proliferation of cancer cells by PCI was shown to be independent of the protease inhibitory action of PCI. Therefore, PCI derivatives with low or no protease inhibitory activity are useful as angiogenesis inhibitors and cancer cell growth inhibitors.
  • Angiogenesis and cell proliferation are important mechanisms in cancer malignancy. Therefore, anticancer agents that act on these mechanisms and inhibit them are useful for preventing cancer malignancy or treating malignant cancers.
  • PCI derivatives that do not have an inhibitory effect on proteases can be expected to reduce the side effects caused by the protease inhibitory action by PCI.

Abstract

Disclosed is an anti-cancer agent comprising a protein C inhibitor (PCI) or a derivative thereof as an active ingredient. The anti-cancer agent has an inhibitory effect on the growth of cancer cells, the metastasis/invasion of cancer and the vascularization. It is revealed that a derivative having a heparin-binding region of a PCI can inhibit the growth of cancer cells, the metastasis of cancer and the vascularization. Thus, the PCI or derivative thereof is useful for the inhibition of the growth and metastasis of cancer and the vascularization.

Description

明 細 書  Specification
プロテイン Cインヒビターを含有する抗癌剤  Anticancer agent containing protein C inhibitor
技術分野  Technical field
[0001] 本発明は、抗癌剤に関する。より具体的には、本発明は、プロテイン Cインヒビター を含有する抗癌剤、プロテイン cインヒビターを含有する抗癌剤による治療方法、およ びプロテイン cインヒビターを含有する抗癌剤の製造方法に関する。  [0001] The present invention relates to an anticancer agent. More specifically, the present invention relates to an anticancer agent containing a protein C inhibitor, a method for treatment with an anticancer agent containing a protein c inhibitor, and a method for producing an anticancer agent containing a protein c inhibitor.
背景技術  Background art
[0002] 近年、先進国における主な死亡原因は、感染症から成人病へとシフトしてきた。こ のような疾病構造の変化の中にあって、特に癌は、多くの国で共通して、主要な死亡 原因の上位を占める重要な疾患である。たとえば日本においては、年間の癌による 死亡者数は 30万人を超えている。この数字は、心疾患による死亡者数のおよそ倍で ある。したがって癌の治療技術の提供は、重要な研究課題である。  [0002] In recent years, the main cause of death in developed countries has shifted from infectious diseases to adult diseases. Amid these changes in disease structure, cancer is an important disease that dominates the leading cause of death, especially in many countries. For example, in Japan, the annual number of deaths from cancer exceeds 300,000. This figure is approximately double the number of deaths from heart disease. Therefore, provision of cancer treatment technology is an important research subject.
[0003] 癌の治療には、一般に、外科的治療、理化学的治療、そして薬物治療等が利用さ れている。理化学的治療とは、放射線療法、粒子線 (荷電重粒子線)治療、あるいは 温熱療法などが含まれる。薬物治療に利用される多くの化学療法剤も実用化された 。更に、ワクチン療法、あるいは細胞免疫療法などの、新しいアプローチについても、 その治療効果が明らかにされつつある。しかし、今なお、癌の治療方法の提供は、重 要な研究課題である。特に、癌の悪性ィ匕を防ぐ技術、あるいは悪性の癌の治療方法 が提供されれば、癌医療に大きく貢献する。  [0003] For the treatment of cancer, surgical treatment, physicochemical treatment, drug treatment and the like are generally used. Physiochemical treatment includes radiation therapy, particle beam (charged heavy particle beam) treatment, or hyperthermia. Many chemotherapeutic agents used for drug treatment have also been put into practical use. Furthermore, the therapeutic effects of new approaches such as vaccine therapy or cellular immunotherapy are being clarified. However, providing cancer treatment methods remains an important research issue. In particular, if a technique for preventing cancer malignancy or a method for treating malignant cancer is provided, it will greatly contribute to cancer medical treatment.
[0004] 一般的に、癌の悪性化のメカニズムは、癌細胞の増殖、浸潤、および転移によって 説明することができる。言い換えれば、増殖した癌細胞が、浸潤によって転移するとき 、その癌は悪性とされる。癌の増殖には、通常、血管新生が伴う。したがって、血管新 生もまた、癌の悪性ィ匕を構成する重要なメカニズムとされて 、る。  [0004] In general, the mechanism of cancer malignancy can be explained by the proliferation, invasion, and metastasis of cancer cells. In other words, when a cancer cell that has proliferated metastasizes by invasion, the cancer is considered malignant. Cancer growth is usually accompanied by angiogenesis. Therefore, angiogenesis is also an important mechanism that constitutes malignant cancer.
[0005] 癌の悪性ィ匕を構成する一連のメカニズムは、一般に次のように説明されて 、る。ま ず原発巣で増殖した癌細胞が原発巣から離脱する。離脱した癌細胞がリンパ液や血 流によって移動し、他の組織に浸潤する。浸潤した癌細胞が再び増殖を開始すると、 転移が成立する。癌細胞の増殖には血管新生が必要である。あるいは原発巣から血 流中への癌細胞の離脱においても、血管新生が重要な意味を持つ。これらの一連の メカニズムの 、ずれかを阻止することができれば、癌の悪性ィ匕を防ぐことができると考 えられている。つまり、癌の治療、とりわけ悪性の癌の治療においては、細胞増殖、浸 潤、転移、そして血管新生が、重要な治療標的となる。 [0005] A series of mechanisms constituting cancer malignancy is generally explained as follows. First, cancer cells that have proliferated in the primary lesion are detached from the primary lesion. The detached cancer cells move by lymph fluid and blood flow and invade other tissues. Metastasis is established when the infiltrated cancer cells begin to grow again. Cancer cell growth requires angiogenesis. Or blood from the primary lesion Angiogenesis also has an important role in the removal of cancer cells into the flow. It is thought that malignant cancer can be prevented if any of these mechanisms can be prevented. In other words, cell growth, infiltration, metastasis, and angiogenesis are important therapeutic targets in the treatment of cancer, especially malignant cancer.
[0006] 癌の 90%以上は上皮細胞で発生する。上皮細胞は相互に E-カドヘリンを介した細 胞間接着によって強固なシート状の構造を保っている。そのため上皮細胞の移動は 制限されている。悪性の癌では、この細胞間の接着機能が弱いために、原発巣から の離脱が起こりやすくなつて 、ると考えられて 、る。  [0006] More than 90% of cancers occur in epithelial cells. Epithelial cells maintain a strong sheet-like structure by intercellular adhesion via E-cadherin. Therefore, the movement of epithelial cells is restricted. In malignant cancers, the adhesion between cells is weak, and it is thought that it is easy to leave the primary lesion.
[0007] し力し細胞の移動性のみでは悪性ィ匕を説明することはできない。通常、固形癌の癌 組織は細胞外基質 (extracellular matrix; ECM)によって包まれている。癌が転移する ためには、癌細胞は、何らかのメカニズムによって ECMを通過しなければならない。 E CMは、主として、次のような成分によって構成されている。 [0007] However, it is not possible to explain malignant diseases only by the mobility of cells. Normally, cancerous tissue of solid cancer is enveloped by an extracellular matrix (ECM). In order for cancer to metastasize, cancer cells must cross the ECM by some mechanism. ECM is mainly composed of the following components.
コラーゲン (collagen) フイブロネクチン (fibronectin)  Collagen Fibronectin
ラミニン (laminin) プロテオグリカン (proteoglycan)  Laminin proteoglycan
エラスチン (elastin)  Elastin
[0008] 各成分、並びにその他の構成成分の割合は、組織によっても相違して 、る。更にこ れらの各成分そのものにも、いくつかのサブタイプが存在する。しかし複数の組織に 共通して、 ECMのもっとも主要な構成成分は、コラーゲンである。悪性の癌は、 ECM のコラーゲンを分解する酵素を産生して組織の中を移動することが明らかにされた。 一般にコラーゲンは安定な分子で、各種のプロテアーゼに対しても耐性を有すること が多い。したがって、プロテアーゼによる ECMの破壊は、癌が悪性ィ匕するための重要 な条件である。 ECMの分解に関与する複数のプロテアーゼが同定されている。 ECM の分解に関与するプロテアーゼは、マトリクスメタ口プロテアーゼ (Matrix metalloprotei nase; MMP)と呼ばれる。 MMPは、癌の転移を防ぐための標的分子として重要視され ている。生体中では、組織メタ口プロテアーゼインヒビター (Tissue inhibitor of metallo proteinase; TIMP)や α - 2-マクログロブリン (Alfa- 2- macroglobulin; 2M)などのプロ テアーゼインヒビターが、 MMPの活性を制御して 、る。  [0008] The ratio of each component, as well as other components, varies depending on the tissue. In addition, there are several subtypes of these components themselves. However, in common with multiple tissues, the most important component of ECM is collagen. Malignant cancer has been shown to move through tissues by producing enzymes that degrade ECM collagen. In general, collagen is a stable molecule and often has resistance to various proteases. Therefore, destruction of ECM by protease is an important condition for cancer to become malignant. Several proteases involved in ECM degradation have been identified. Proteases involved in ECM degradation are called matrix metalloprotease (MMP). MMP is regarded as an important target molecule for preventing cancer metastasis. In vivo, protease inhibitors such as tissue inhibitor of metallo proteinase (TIMP) and α-2-macroglobulin (Alfa-2- macroglobulin; 2M) regulate MMP activity. .
[0009] 一方、プロテアーゼである MMPの中には、他のプロテアーゼによる消化によって、 潜在型力 活性型に変化するものがある。たとえば MMP-1や MMP-3は、プラスミンに よって活性型となることが知られている。プラスミンは、プラスミノーゲンの活性ィ匕によ つてプロテアーゼ活性を獲得する。そしてプラスミノーゲンの活性ィ匕に必要なのが、 プラスミノーゲンァクチベータ (plasminogen activator; PA)である。 PAにはゥロキナー ゼ型 (uPA)と組織型 (tPA)とが存在する。 tPAは主に線溶系に作用し、血栓溶解剤とし ても利用されて 、る分子である。一方 uPAは癌の浸潤や転移に関与する可能性が指 摘されている。 [0009] On the other hand, in MMP, which is a protease, by digestion with other proteases, Potential power Some change to active form. For example, MMP-1 and MMP-3 are known to be activated by plasmin. Plasmin acquires protease activity through the activity of plasminogen. What is necessary for the activity of plasminogen is a plasminogen activator (PA). There are urokinase type (uPA) and tissue type (tPA) in PA. tPA is a molecule that acts mainly on the fibrinolytic system and is also used as a thrombolytic agent. On the other hand, it has been pointed out that uPA may be involved in cancer invasion and metastasis.
[0010] 血管新生が癌の悪性ィ匕を構成する重要なメカニズムであることは既に述べた。した がって、 MMPと同様に、癌組織における血管新生も、癌の治療戦略の重要な標的と 考えられている。実際、血管内皮成長因子 (vascular endothelial growth factor; VEG F)の発現が、多くの癌において亢進していることが知られている。また ECMを分解す るプロテアーゼは、先に述べた細胞の移動のみならず、血管新生においても重要な 働きを持つことが指摘されている。たとえばプロテアーゼは、 ECMを分解して血管を 新生するためのスペースを作る。このように、癌の悪性化には、さまざまなプロテア一 ゼの活性制御システムが複雑に関与して 、る。  [0010] It has already been described that angiogenesis is an important mechanism that constitutes malignant cancer. Therefore, as with MMPs, angiogenesis in cancer tissues is considered an important target for cancer treatment strategies. In fact, it is known that the expression of vascular endothelial growth factor (VEG F) is increased in many cancers. In addition, it has been pointed out that proteases that degrade ECM have an important role not only in the above-mentioned cell migration but also in angiogenesis. Proteases, for example, create space for breaking down ECM and creating blood vessels. Thus, the malignant transformation of cancer involves intricately involved in various protease activity control systems.
[0011] 一般にプロテアーゼは、生体中においてはそのインヒビターとの結合によって活性 を制御されている。また PAのようなプロテアーゼ活性ィ匕因子の中にも、インヒビターで その機能が制御されているものもある。たとえば、先に述べた uPAは、尿中ではプロテ イン Cインヒビター (protein C inhibitor; PCI)と複合体を形成していることが明らかにさ れている（非特許文献 1)。また PCIが uPAの作用を阻害することも知られている（非特 許文献 2)。  [0011] In general, the activity of protease is controlled in the living body by binding to its inhibitor. Some protease activity factors such as PA are controlled by inhibitors. For example, uPA described above has been shown to form a complex with protein C inhibitor (PCI) in urine (Non-patent Document 1). PCI is also known to inhibit the action of uPA (Non-Patent Document 2).
PCIは、抗凝固系のプロテアーゼであるプロテイン Cの阻害因子として同定されたプ 口テアーゼインヒビターである（非特許文献 3、非特許文献 4)。セリンプロテアーゼ (SE RPIN)ファミリーに属し、トロンビン、 Xa因子、 XIa因子、血漿カリクレイン、そして uPA等 に対する阻害作用を有することが確認されている。 PAのインヒビターとして同定され たプラスミノーゲンァクチべ一タインヒビタ^—— 3(plasminogen activator inhibitor 3)は 、 PCIと同じ分子である。  PCI is a protease inhibitor identified as an inhibitor of protein C, an anticoagulant protease (Non-patent Document 3, Non-patent Document 4). It belongs to the serine protease (SE RPIN) family and has been confirmed to have an inhibitory action on thrombin, factor Xa, factor XIa, plasma kallikrein, and uPA. Plasminogen activator inhibitor 3 identified as an inhibitor of PA (plasminogen activator inhibitor 3) is the same molecule as PCI.
[0012] 非特許文献 1： Stump D.C. et al, J. Biol. Chem. 261:12759-66, 1986 非特許文献 2 : Stief T.W. et al.， Biol. Chem. Hoppe Seyler 368:1427-33, 1987 非特許文献 3 :Marlar R.A. et al, J. Clin. Invest. 66:1186-9, 1980 [0012] Non-Patent Document 1: Stump DC et al, J. Biol. Chem. 261: 12759-66, 1986 Non-patent document 2: Stief TW et al., Biol. Chem. Hoppe Seyler 368: 1427-33, 1987 Non-patent document 3: Marlar RA et al, J. Clin. Invest. 66: 1186-9, 1980
非特許文献 4: Suzuki K. et al" J. Biol. Chem. 258:163-8, 1983  Non-Patent Document 4: Suzuki K. et al "J. Biol. Chem. 258: 163-8, 1983
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0013] 本発明は、抗癌剤の提供を課題とする。あるいは本発明は、癌の細胞増殖、転移、 および血管新生の抑制作用を有する抗癌剤の提供を課題とする。また、本発明は、 抗癌剤による治療方法および抗癌剤の製造方法に関する。 [0013] An object of the present invention is to provide an anticancer agent. Alternatively, an object of the present invention is to provide an anticancer agent having an inhibitory action on cancer cell proliferation, metastasis, and angiogenesis. The present invention also relates to a method for treatment with an anticancer agent and a method for producing an anticancer agent.
課題を解決するための手段  Means for solving the problem
[0014] 本発明者は、 PCIが腎癌組織で発現が著しく低下していた事実に基づき、 PCIの発 現低下が腎癌の発生、腎癌細胞の増殖と関係すると考え、 PCIの腎癌細胞の浸潤転 移に及ぼす効果を検討した。そして、 PCIが腎癌細胞のマトリゲル (in vitro)における 浸潤を阻害することを既に明らかにしている (Wakita T. et al., Int. J. Cancer 108:516 -23, 2004)。また PCIを過剰発現させた乳癌細胞において、細胞接着分子が増加す ることが報告されている (Palmieri D. et al., J. Biol. Chem. 277:40950-7, 2002)。これ らの PCIの癌細胞に対する作用を in vivoにおいて確認するために、本発明者らは腎 癌細胞 CaW-1の増殖と転移に及ぼす PCIの影響を観察した。ところが実験に用いた C aki- 1は、 SCIDマウス中に生着しなかった (Wakita T. et al., Int. J. Cancer 108:516-2 3, 2004)。そのため、実際に生体中 (in vivo)において、 PCIが腎癌に対してどのような 作用を有するのかは、現在もなお明らかでない。  [0014] Based on the fact that the expression of PCI was significantly decreased in renal cancer tissues, the present inventor considered that the decrease in the expression of PCI is related to the occurrence of renal cancer and the proliferation of renal cancer cells. The effect on cell invasive transition was examined. It has already been shown that PCI inhibits invasion of renal cancer cells in Matrigel (in vitro) (Wakita T. et al., Int. J. Cancer 108: 516 -23, 2004). In addition, cell adhesion molecules have been reported to increase in breast cancer cells overexpressing PCI (Palmieri D. et al., J. Biol. Chem. 277: 40950-7, 2002). In order to confirm the effect of these PCIs on cancer cells in vivo, the present inventors observed the effect of PCI on the proliferation and metastasis of renal cancer cells CaW-1. However, Caki-1 used in the experiment did not engraft in SCID mice (Wakita T. et al., Int. J. Cancer 108: 516-2 3, 2004). For this reason, it is still unclear what effect PCI has on kidney cancer in vivo.
[0015] そこで本発明者らは、 Caki-1細胞に代えて乳癌細胞 MDA-231細胞をもちいて検討 を行い、生体内において PCIが癌細胞に与える影響を観察することに成功した。そし て PCIが生体内において癌細胞の増殖や転移を阻害する作用を持つことを明らかに して本発明を完成した。更に、 PCIの in vivoでの抗癌作用の一部は、 PCIのプロテア ーゼ阻害作用に依存しないことを見出し、本発明を完成した。すなわち本発明は、以 下の抗癌剤を提供するものである：  [0015] Therefore, the present inventors have examined using breast cancer cells MDA-231 cells instead of Caki-1 cells, and succeeded in observing the effect of PCI on cancer cells in vivo. The present invention was completed by clarifying that PCI has the action of inhibiting the growth and metastasis of cancer cells in vivo. Furthermore, the present inventors have found that a part of PCI's in vivo anticancer activity does not depend on PCI's protease inhibitory action, thus completing the present invention. That is, the present invention provides the following anticancer agents:
〔1〕プロテイン Cインヒビター (PCI)またはその誘導体を有効成分として含有する抗癌 剤。 〔2〕癌の増殖、癌の転移、および血管新生力 選択された活性の少なくとも 1つを抑 制する〔1〕に記載の抗癌剤。 [1] An anticancer agent comprising a protein C inhibitor (PCI) or a derivative thereof as an active ingredient. [2] The anticancer agent according to [1], which suppresses at least one selected activity of cancer growth, cancer metastasis, and angiogenesis.
〔3〕癌が乳癌である〔1〕または〔2〕に記載の抗癌剤。  [3] The anticancer agent according to [1] or [2], wherein the cancer is breast cancer.
〔4〕プロテイン Cインヒビターの誘導体のプロテアーゼ阻害作用がプロテイン Cインヒ ビターよりも低い〔2〕に記載の抗癌剤。  [4] The anticancer agent according to [2], wherein the protein C inhibitor derivative has a protease inhibitory action lower than that of the protein C inhibitor.
〔5〕プロテイン Cインヒビターの誘導体力 プロテイン Cインヒビターのへパリン結合領 域を含む蛋白質である〔4〕に記載の抗癌剤。  [5] Derivative power of protein C inhibitor The anticancer agent according to [4], which is a protein containing a heparin-binding region of a protein C inhibitor.
〔6〕プロテイン Cインヒビターの誘導体力 へパリンおよびへパリン様グリコサミノダル カンのいずれかまたは両方との結合能を有する蛋白質である〔5〕に記載の抗癌剤。 〔7〕癌の浸潤を抑制する〔1〕に記載の抗癌剤。  [6] Derivative power of protein C inhibitor The anticancer agent according to [5], which is a protein capable of binding to either or both of heparin and heparin-like glycosaminodalkane. [7] The anticancer agent according to [1], which suppresses cancer infiltration.
〔8〕プロテイン Cインヒビターの誘導体がプロテアーゼ阻害作用を有する〔7〕に記載 の抗癌剤。  [8] The anticancer agent according to [7], wherein the protein C inhibitor derivative has a protease inhibitory action.
あるいは本発明は、プロテイン Cインヒビターまたはその誘導体の抗癌剤の製造に おける使用に関する。また本発明は、プロテイン Cインヒビターまたはその誘導体の癌 の治療における使用に関する。加えて本発明は、プロテイン Cインヒビターまたはそ の誘導体を投与する工程を含む、癌の治療方法に関する。すなわち、本発明は、以 下を提供するものである：  Alternatively, the present invention relates to the use of a protein C inhibitor or a derivative thereof in the production of an anticancer agent. The invention also relates to the use of protein C inhibitors or derivatives thereof in the treatment of cancer. In addition, the present invention relates to a method for treating cancer comprising administering a protein C inhibitor or a derivative thereof. That is, the present invention provides the following:
〔9〕プロテイン Cインヒビター (PCI)またはその誘導体を投与する工程を含む、癌の治 療方法。  [9] A method for treating cancer, comprising a step of administering a protein C inhibitor (PCI) or a derivative thereof.
〔10〕癌の増殖、癌の転移、および血管新生力 選択された活性の少なくとも 1つが 抑制される、〔9〕に記載の方法。  [10] The method according to [9], wherein at least one of selected activities of cancer growth, cancer metastasis, and angiogenic activity is suppressed.
〔11〕癌が乳癌である〔9〕または〔10〕に記載の方法。  [11] The method according to [9] or [10], wherein the cancer is breast cancer.
〔 12〕プロテイン Cインヒビターの誘導体のプロテアーゼ阻害作用がプロテイン Cインヒ ビターよりも低い、〔10〕に記載の方法。  [12] The method according to [10], wherein the protein C inhibitor derivative has a protease inhibitory action lower than that of the protein C inhibitor.
〔13〕プロテイン Cインヒビターの誘導体力 プロテイン Cインヒビターのへパリン結合 領域を含む蛋白質である、〔12〕に記載の方法。  [13] Derivative power of protein C inhibitor The method according to [12], which is a protein containing a heparin-binding region of a protein C inhibitor.
〔14〕プロテイン Cインヒビターの誘導体力 へパリンおよびへパリン様グリコサミノダル カンのいずれかまたは両方との結合能を有する蛋白質である、〔13〕に記載の方法。 〔15〕癌の浸潤が抑制される、〔9〕に記載の方法。 [14] Derivative power of protein C inhibitor [13] The method according to [13], which is a protein capable of binding to either or both of heparin and heparin-like glycosaminodalkane. [15] The method according to [9], wherein cancer infiltration is suppressed.
〔16〕プロテイン Cインヒビターの誘導体がプロテアーゼ阻害作用を有する、 [15]に 記載の方法。  [16] The method according to [15], wherein the protein C inhibitor derivative has a protease inhibitory action.
〔17〕プロテイン Cインヒビターまたはその誘導体を有効成分として含有する抗癌剤の 製造のための使用。  [17] Use for the manufacture of an anticancer agent containing a protein C inhibitor or a derivative thereof as an active ingredient.
〔18〕抗癌剤が、癌の増殖、癌の転移、および血管新生力 選択された活性の少なく とも 1つを抑制する、〔17〕に記載の使用。  [18] The use according to [17], wherein the anticancer agent suppresses at least one of the selected activity of cancer growth, cancer metastasis, and angiogenic potential.
〔19〕癌が乳癌である、〔 17〕または〔 18〕に記載の使用。  [19] Use according to [17] or [18], wherein the cancer is breast cancer.
〔20〕プロテイン Cインヒビターの誘導体のプロテアーゼ阻害作用がプロテイン Cインヒ ビターよりも低い、〔18〕に記載の使用。  [20] The use according to [18], wherein the protein C inhibitor derivative has a protease inhibitory action lower than that of the protein C inhibitor.
〔21〕プロテイン Cインヒビターの誘導体力 プロテイン Cインヒビターのへパリン結合 領域を含む蛋白質である、〔20〕に記載の使用。  [21] Derivative power of protein C inhibitor Use according to [20], which is a protein containing a heparin-binding region of a protein C inhibitor.
[22]プロテイン Cインヒビターの誘導体力 へパリンおよびへパリン様グリコサミノダル カンのいずれかまたは両方との結合能を有する蛋白質である、〔21〕に記載の使用。 〔23〕抗癌剤が、癌の浸潤を抑制する、〔17〕に記載の使用。  [22] Derivative power of protein C inhibitor Use according to [21], which is a protein capable of binding to either or both of heparin and heparin-like glycosaminodalkane. [23] The use according to [17], wherein the anticancer agent suppresses cancer invasion.
〔24〕プロテイン Cインヒビターの誘導体がプロテアーゼ阻害作用を有する、〔23〕に 記載の使用。 [24] The use according to [23], wherein the protein C inhibitor derivative has a protease inhibitory action.
あるいは本発明は、 PCIまたはその誘導体を有効成分として含有する、癌細胞の増 殖抑制剤、癌の転移抑制剤、癌の浸潤阻害剤、あるいは癌組織における血管新生 の阻害剤を提供する。また本発明は、 PCほたはその誘導体の、癌細胞の増殖抑制 剤、癌の転移抑制剤、癌の浸潤阻害剤、あるいは癌組織における血管新生の阻害 剤の製造における使用に関する。  Alternatively, the present invention provides a cancer cell growth inhibitor, a cancer metastasis inhibitor, a cancer invasion inhibitor, or an angiogenesis inhibitor in cancer tissue, comprising PCI or a derivative thereof as an active ingredient. The present invention also relates to the use of PC or a derivative thereof in the manufacture of a cancer cell growth inhibitor, a cancer metastasis inhibitor, a cancer invasion inhibitor, or an angiogenesis inhibitor in cancer tissue.
加えて本発明は、 PCほたはその誘導体の、癌細胞の増殖抑制、癌の転移抑制、 癌の浸潤阻害、あるいは癌組織における血管新生の阻害における使用に関する。更 に本発明は、 PCほたはその誘導体を投与する工程を含む、癌細胞の増殖抑制方法 In addition, the present invention relates to the use of PC or a derivative thereof in inhibiting cancer cell growth, inhibiting cancer metastasis, inhibiting cancer invasion, or inhibiting angiogenesis in cancer tissue. Furthermore, the present invention provides a method for inhibiting the growth of cancer cells, comprising the step of administering PC or a derivative thereof.
、癌の転移抑制方法、癌の浸潤阻害方法、あるいは癌組織における血管新生の阻 害方法に関する。 The present invention also relates to a method for inhibiting cancer metastasis, a method for inhibiting cancer invasion, or a method for inhibiting angiogenesis in cancer tissue.
発明の効果 [0018] 本発明により PCIまたはその誘導体を有効成分として含有する抗癌剤が提供された 。本発明によって、 PCIはそのプロテアーゼ阻害作用依存的に、癌の浸潤を抑制する ことが明らかにされた。したがって、プロテアーゼ阻害作用を有する PCI誘導体は、癌 の浸潤の抑制剤として有用である。一方、 PCIによる癌における血管新生阻害作用は 、 PCIのプロテアーゼ阻害作用に依存しな力つた。同様に PCIによる癌細胞の増殖阻 害作用も、 PCIのプロテアーゼ阻害作用に依存しないことが示された。したがって、プ 口テアーゼ阻害作用が低い、あるいは欠失した PCI誘導体は、血管新生の阻害剤並 びに癌細胞の増殖抑制剤として有用である。 PCIのプロテアーゼ阻害作用を欠失し た誘導体が血管新生や細胞増殖を抑制することは、本発明によって示された新規な 知見である。 The invention's effect [0018] The present invention provides an anticancer agent containing PCI or a derivative thereof as an active ingredient. According to the present invention, it has been clarified that PCI suppresses cancer invasion depending on its protease inhibitory action. Therefore, PCI derivatives having protease inhibitory action are useful as inhibitors of cancer invasion. On the other hand, the angiogenesis inhibitory effect in cancer by PCI was dependent on PCI protease inhibitory action. Similarly, the inhibition of proliferation of cancer cells by PCI was shown to be independent of the protease inhibitory action of PCI. Therefore, PCI derivatives with low or no protease inhibitory activity are useful as angiogenesis inhibitors and cancer cell growth inhibitors. It is a novel finding demonstrated by the present invention that a derivative lacking the protease inhibitory action of PCI suppresses angiogenesis and cell proliferation.
[0019] プロテアーゼ阻害作用が低い、あるいは欠失した PCI誘導体の使用によって、プロ テアーゼ阻害作用に起因する副作用を避けることができる。たとえば PCIは、線溶系 に対する強力な阻害作用を有する。したがって PCIの投与によって、線溶系の阻害が 起きる心配がある。しかし本発明に基づ!/、てプロテアーゼ阻害作用を欠失した PCI誘 導体を利用すれば、抗癌作用を維持しながら、線溶系に対する阻害作用の無い薬 剤とすることができる。  [0019] By using a PCI derivative having a low protease inhibitory action or a deletion, side effects caused by the protease inhibitory action can be avoided. PCI, for example, has a strong inhibitory effect on the fibrinolytic system. Therefore, there is a concern that the administration of PCI may cause inhibition of the fibrinolytic system. However, based on the present invention, if a PCI derivative lacking protease inhibitory action is used, a drug having no inhibitory action on the fibrinolytic system can be obtained while maintaining the anticancer action.
[0020] また、 PCIは、抗凝固作用ゃ抗炎症作用を有する活性化プロテイン C(APC)を阻害 し、生体内の凝固制御系，炎症制御系を阻害する恐れがあり、同様に、 PCIは、抗凝 固作用を発揮するトロンビン'トロンボモジュリン複合体を阻害してプロテイン Cの APC への変換を阻害し、 APCの生成を低下させる恐れがある。しかし、プロテアーゼ阻害 作用を欠失した PCI誘導体を使用すればそれらの心配はなくなり、抗癌作用を維持し ながら、凝固制御系 ·炎症制御系に対して影響を与えな 、抗癌剤とすることができる  [0020] In addition, PCI inhibits activated protein C (APC) having anti-coagulant action and anti-inflammatory action, and may inhibit coagulation control system and inflammation control system in vivo. Inhibiting thrombin 'thrombomodulin complex, which exerts anti-coagulant action, inhibits the conversion of protein C to APC and may reduce APC production. However, if PCI derivatives lacking protease inhibitory action are used, those concerns are eliminated, and the anticoagulant control system and inflammation control system are not affected while maintaining anticancer action, and can be used as an anticancer drug.
[0021] 更に、アンジォスタチンやエンドスタチンなどの血管新生阻害作用を有する内因性 の蛋白質の存在が知られている。これらの内因性血管新生阻害物質は、強力な血管 新生阻害作用を有する。その作用を利用して、癌の血管新生作用を標的とする抗癌 剤の開発が進められている。これらの内因性の血管新生阻害因子は、プラスミンゃコ ラーゲンがある種のプロテアーゼによって消化されて産生されると考えられて 、る。プ 口テアーゼ阻害剤は抗癌剤としての作用を有するとともに、一方では、これらの血管 新生阻害因子の産生を妨げる可能性を伴っている。本発明によれば、プロテアーゼ 阻害作用を伴わない薬剤を提供することができる。すなわち本発明によれば、内因 性の血管新生阻害物質の産生に影響しない抗癌剤が提供される。 [0021] Furthermore, the existence of endogenous proteins having angiogenesis inhibitory action such as angiostatin and endostatin is known. These endogenous angiogenesis inhibitors have a strong angiogenesis inhibitory action. Utilizing this action, development of anticancer agents targeting the angiogenic action of cancer has been underway. These endogenous angiogenesis inhibitors are thought to be produced by digestion of plasmin collagen by certain proteases. The Oral thease inhibitors act as anticancer agents, but on the other hand have the potential to prevent the production of these angiogenesis inhibitors. ADVANTAGE OF THE INVENTION According to this invention, the chemical | medical agent without a protease inhibitory effect can be provided. That is, according to the present invention, an anticancer agent that does not affect the production of an endogenous angiogenesis inhibitor is provided.
[0022] へパリン結合性のセルピン (SERPIN)であるカリスタチン (Kallistatin)力 血管新生を 伴う癌細胞の増殖に対して抑制作用を持つことが明らかにされている (Miao R.Q. et a 1., Blood 100:3245-52, 2002)。カリスタチンによる癌細胞の血管新生の阻害作用は、 VEGFとその受容体との結合において重要なへパリンとの結合阻害によるものと推定 された (Miao R.Q. et al.， Am. J. Physiol. Cell Physiol. 284:C1604- 13, 2003)。同様に へノ^ン結合性セルピンであるアンチトロンビン (AT)も血管新生阻害作用を持つこと が知られている (O'Reilly MS et al.， Science 285:1926-8, 1999)。本発明では、これら の報告とは異なる機構、すなわち、 PCIのプロテアーゼ阻害作用に依存しない機構に よる抗癌作用が確認された。 PCIのプロテアーゼ阻害作用非依存性の抗癌作用は、 既知の知見からは予測できなつた作用である。 [0022] Kallistatin, a heparin-binding serpin (SERPIN), has been shown to have an inhibitory effect on the growth of cancer cells with angiogenesis (Miao RQ et a 1., Blood 100: 3245-52, 2002). Inhibition of angiogenesis of cancer cells by calistatin was presumed to be due to inhibition of heparin binding, which is important in binding VEGF to its receptor (Miao RQ et al., Am. J. Physiol. Cell Physiol 284: C1604- 13, 2003). Similarly, anti-thrombin (AT), a hedon-binding serpin, is known to have an angiogenesis inhibitory effect (O'Reilly MS et al., Science 285: 1926-8, 1999). In the present invention, an anticancer effect by a mechanism different from these reports, that is, a mechanism independent of the protease inhibitory action of PCI was confirmed. PCI's protease inhibitor-independent anticancer effect is an action that could not be predicted from known findings.
図面の簡単な説明  Brief Description of Drawings
[0023] [図 1]組換え原型 PCI、 R354APCI、および degPCIのウェスタンブロット解析を示す写 真である。 各糸且換えタンパク質（レーン 1、原型 PCI ;レーン 2、 R354APCI ;レーン 3、 d egPCl) 1 μ gを、 SDS- PAGE、ならびに抗ヒト PCIゥサギ IgGおよび次にアルカリホスファ ターゼ結合抗ゥサギ IgGを用いたウェスタンプロットに供した。材料および方法に記載 したように、ウェスタンブルー安定ィ匕基質を用いて各バンドを可視化した。  [0023] [Fig. 1] Photograph showing Western blot analysis of recombinant prototype PCI, R354APCI, and degPCI. 1 μg of each thread-replaceable protein (lane 1, prototype PCI; lane 2, R354APCI; lane 3, d egPCl), SDS-PAGE, and anti-human PCI rabbit IgG and then alkaline phosphatase-conjugated anti-rabbit IgG It used for the Western plot used. Each band was visualized using a Western Blue stable substrate as described in Materials and Methods.
[図 2]組換え原型 PCI、 R354APCI、および degPCIによる APCの阻害を示すグラフであ る。 ヒト APCに対する各組換え PCIの阻害活性を以下のように決定した。へパリン（10 U/ml)の存在下において、各組換えタンパク質 (40 g/ml)をヒト APC(40 /z g/ml)と共 に 20分間インキュベートした後、 S-2366を用いて残存 APC活性を決定した。  FIG. 2 is a graph showing inhibition of APC by recombinant prototype PCI, R354APCI, and degPCI. The inhibitory activity of each recombinant PCI against human APC was determined as follows. Each recombinant protein (40 g / ml) was incubated with human APC (40 / zg / ml) for 20 minutes in the presence of heparin (10 U / ml), and then the remaining APC was used with S-2366. Activity was determined.
[図 3A]トランスフ クシヨンして!/、な!/、MDA-231細胞の浸潤活性に及ぼす PCIの効果 を示すグラフである。 図 3A— Dは、トランスフエクシヨンしていない MDA-231細胞（2 X 105)を、様々な濃度の PCI (A)、および抗ヒト uPA IgG (B)、 PAI- 1 (C)、または uPA ( D)の存在下で DMEM 500 μ 1中に懸濁し、培養インサート（上チャンバ一）に入れた。 下チャンバ一は、化学誘引物質としての 10% FBSを含む DMEM 750 1を含んだ。 24 時間インキュベートした後、膜の下表面上の浸潤細胞を固定し染色した。光学顕微 鏡下で 100 X倍率において、細胞をカウントした。データは、 4枚の独立した浸潤膜に 由来する細胞の数 (平均値士 S.D.)として表す。 NDは「未検」を示す。 FIG. 3A is a graph showing the effect of PCI on invasive activity of transduced! /, NA! /, MDA-231 cells. Figure 3A-D shows untransfected MDA-231 cells (2 x 10 5 ), various concentrations of PCI (A), and anti-human uPA IgG (B), PAI-1 (C), or Suspended in 500 μl of DMEM in the presence of uPA (D) and placed in the culture insert (upper chamber 1). The lower chamber contained DMEM 750 1 containing 10% FBS as a chemoattractant. After 24 hours of incubation, infiltrating cells on the lower surface of the membrane were fixed and stained. Cells were counted at 100X magnification under an optical microscope. Data are expressed as the number of cells derived from 4 independent infiltrating membranes (mean SD). ND indicates “not tested”.
[図 3B]トランスフエクシヨンして!/、な!/、MDA-231細胞の浸潤活性に及ぼす抗ヒト uPA I gGの効果を示すグラフである。 FIG. 3B is a graph showing the effect of anti-human uPA IgG on the invasive activity of MDA-231 cells after transfection! /, NA! /.
[図 3C]トランスフエクシヨンしていない MDA-231細胞の浸潤活性に及ぼす PAI-1の効 果を示すグラフである。  FIG. 3C is a graph showing the effect of PAI-1 on the invasion activity of non-transfected MDA-231 cells.
[図 3D]トランスフエクシヨンして!/ヽな 、MDA-231細胞の浸潤活性に及ぼす uPAの効果 を示すグラフである。  FIG. 3D is a graph showing the effect of uPA on the invasive activity of MDA-231 cells after transformation!
[図 4]MDA-231細胞浸潤に及ぼす組換え原型 PCI、 R354APCI、および degPCIの効 果を示すグラフである。トランスフエクシヨンしていない MDA-231細胞（2 X 105)を、 10 μ g/mlの原型 PCI、 R354APCI、または degPCIの存在下で DMEM 500 μ 1中に懸濁し 、培養インサート（上チャンバ一）に入れた。下チャンバ一は、化学誘引物質としての 10% FBSを含む DMEM 750 1を含んだ。 24時間インキュベートした後、膜の下表面上 の浸潤細胞を固定し染色した。光学顕微鏡下で 100 X倍率において、細胞をカウント した。データは、 4枚の独立した浸潤膜に由来する細胞の数 (平均値士 S.D.)として表 す。 *p〈0.05。 FIG. 4 is a graph showing the effects of recombinant prototype PCI, R354APCI, and degPCI on MDA-231 cell invasion. Untransfected MDA-231 cells (2 x 10 5 ) were suspended in DMEM 500 μl in the presence of 10 μg / ml prototypical PCI, R354APCI, or degPCI and the culture insert (upper chamber ). The lower chamber contained DMEM 750 1 containing 10% FBS as a chemoattractant. After 24 hours of incubation, infiltrating cells on the lower surface of the membrane were fixed and stained. Cells were counted at 100X magnification under a light microscope. Data are expressed as the number of cells from 4 independent invasive membranes (mean SD). * p <0.05.
[図 5]原型 PCI、 R354APCI、および degPCI発現 MDA-231細胞株の浸潤活性を示す グラフである。 MDA-PCI、 MDA-R354APCI, MDA-degPCI、または MDA-Mock細胞 株（2 X 105)を DMEM 500 μ 1中に懸濁し、培養インサート（上チャンバ一）に入れた。 その後の実験手順は、図 4の説明に記載したものと基本的に同じである。データは、 4 枚の独立した浸潤膜に由来する細胞の数 (平均値士 S.D.)として表す。 *ρく 0.05。 FIG. 5 is a graph showing the invasion activity of prototype PCI, R354APCI, and degPCI-expressing MDA-231 cell lines. MDA-PCI, MDA-R354APCI, MDA-degPCI, or MDA-Mock cell line (2 × 10 5 ) was suspended in DMEM 500 μl and placed in a culture insert (upper chamber 1). The subsequent experimental procedure is basically the same as that described in the explanation of FIG. Data are expressed as the number of cells derived from 4 independent infiltrating membranes (mean SD). * ρ く 0.05.
[図 6]SCIDマウスにおける原型 PCI、 R354APCUおよび degPCI発現 MDA-231細胞株 の増殖を示すグラフである。滅菌した DMEM 200 μ 1中の 2 χ 105個細胞の MDA-PCI、 MDA- R354APCI、 MDA- degPCI、または MDA- Mockを、 5週齢の雄 SCIDマウスの腹 壁に皮内注射し、ノギス測定 (calipation)により腫瘍容積を測定した。 FIG. 6 is a graph showing the growth of prototype PCI, R354APCU and degPCI expressing MDA-231 cell lines in SCID mice. Two χ 10 5 cells of MDA-PCI, MDA-R354APCI, MDA-degPCI, or MDA-Mock in sterile DMEM 200 μl are injected intradermally into the abdominal wall of 5-week-old male SCID mice, and calipers are used. Tumor volume was measured by calipation.
[図 7]SCIDマウスにおける原型 PCI、 R354APCUおよび degPCI発現 MDA-231細胞株 の実験的転移を示すグラフ、及び写真である。 MDA-PCU MDA-R354APCU MDA-d egPCI、または MDA- Mock細胞株（2 x 105)を DMEM 200 1に懸濁し、 5週齢の雌 SCI Dマウスに静脈内注射した。 35日後に肺を摘出して固定し、腫瘍巣数をカウントする ことにより肺の転移形成を定量した。代表的な結果を示す写真 (A)、および 6匹のマウ スの増殖巣数 (平均値士 S.D.)として表したグラフ (B)を示す。 [Fig. 7] Prototype PCI, R354APCU and degPCI expressing MDA-231 cell line in SCID mice 2 is a graph and a photograph showing the experimental transition. MDA-PCU MDA-R354APCU MDA-d egPCI, or MDA-Mock cell line (2 × 10 5 ) was suspended in DMEM 200 1 and injected intravenously into 5-week-old female SCI D mice. After 35 days, the lungs were removed and fixed, and the number of tumors was counted to quantify the formation of lung metastases. A photograph (A) showing a typical result and a graph (B) expressed as the number of growth foci (mean value SD) of 6 mice are shown.
[図 8]SCIDマウスにおける、原型 PCI、 R354APCUおよび degPCI発現 MDA-231細胞 株を含むマトリゲルでの新血管形成および血管新生の定量的解析を示すグラフ、及 び写真である。 VEGFおよび様々な濃度のへパリンと共に MDA- PCI、 MDA- R354AP CI、 MDA-degPCUまたは MDA- Mock (2 x 106細胞）を含むマトリゲル 0.5 mlを、各雄 SCIDマウスの腹部正中近傍に皮下注射した。 3日後、デジタルカメラシステム（Olymp us,ニューヨーク州、メルビル)で、マトリゲルへの新血管形成を撮影した (A)。同時に 、マトリゲルプラグを除去し、ハンクス液に溶解した 0.1%コラゲナーゼ 1 mlでこれを消 化し、ヘモグロビンアツセィ法により新血管形成を定量した。それぞれについて、 n〉6 および平均値士 S.Dを示す (B)。 FIG. 8 is a graph and a photograph showing a quantitative analysis of neovascularization and neovascularization in Matrigel containing prototype PCI, R354APCU and degPCI-expressing MDA-231 cell lines in SCID mice. Subcutaneous injection of 0.5 ml Matrigel containing MDA-PCI, MDA-R354AP CI, MDA-degPCU or MDA-Mock (2 x 10 6 cells) with VEGF and various concentrations of heparin near the midline of the abdomen of each male SCID mouse did. Three days later, a digital camera system (Olymp us, Melville, NY) was used to photograph neovascularization in Matrigel (A). At the same time, the Matrigel plug was removed, and it was extinguished with 1 ml of 0.1% collagenase dissolved in Hanks' solution, and the formation of new blood vessels was quantified by the hemoglobin assay. For each, n> 6 and mean value SD are shown (B).
[図 9]CAMでの血管形成に及ぼす原型 PCI、 R354APCI、および degPCの効果を示す グラフである。原型 PCI、 R354APCUおよび degPCIを含むディスクを、胚の尿漿膜 (C AM)上に載せた。デジタルカメラシステム（01ympus、ニューヨーク州、メルビル）およ び NIH Image 1.61 (NIH、メリーランド州、ベテスダ）により、 CAMの画像を撮影した。 新血管長を画素として測定し、結果を平均値士 S.Dとして表す。 *pく 0.05。  FIG. 9 is a graph showing the effects of prototype PCI, R354APCI, and degPC on angiogenesis in CAM. Discs containing prototype PCI, R354APCU and degPCI were mounted on embryonic serosa (CAM). CAM images were taken with a digital camera system (01ympus, Melville, NY) and NIH Image 1.61 (NIH, Bethesda, MD). The new blood vessel length is measured as a pixel, and the result is expressed as a mean value S.D. * p <0.05.
[図 10]HUVECの管形成に及ぼす原型 PCI、 R354APCUおよび degPCIの効果を示す グラフ、及び写真である。マトリゲルをコーティングした 24ゥエルプレート上で、 5% FBS を添カ卩した MCDB— 131中、 HUVEC (2 x 104細胞）を、 10 gの原型 PCI、 R354APCIも しくは degPCIの存在下、またはこれらの PCIの非存在下（対照）において培養した。プ レーティングしてから 6時間後に写真を撮影し (倍率 X 100) (A)、管形成に及ぼす様々 な組換え変異体 PCIの効果を定量解析した (B)。管長を画素として測定し、結果を平 均値士 S.Dとして表す。 *p〈0.05。 FIG. 10 is a graph and a photograph showing the effects of prototype PCI, R354APCU and degPCI on HUVEC tube formation. HUVEC (2 x 10 4 cells) in MCDB-131 supplemented with 5% FBS on a 24well plate coated with Matrigel in the presence of 10 g of prototype PCI, R354APCI or degPCI, or These were cultured in the absence of PCI (control). Photographs were taken 6 hours after plating (magnification X 100) (A) and quantitative analysis of the effects of various recombinant mutant PCIs on tube formation (B). The tube length is measured in pixels, and the result is expressed as an averager SD. * p <0.05.
発明を実施するための最良の形態 BEST MODE FOR CARRYING OUT THE INVENTION
本発明により、 PCIが in vivoにおいて抗癌作用を示すことが見出された。 PCIによる 抗癌作用は、癌細胞の増殖、浸潤、そして血管新生の抑制によってもたらされること が確認された。更に、これらの抗癌作用を維持した複数の PCI誘導体の存在が明ら 力にされた。これらの知見に基づいて、 PCIまたはその誘導体を有効成分として含有 する、抗癌剤が提供された。 According to the present invention, it has been found that PCI exhibits an anticancer effect in vivo. By PCI It was confirmed that the anticancer effect is brought about by suppression of cancer cell proliferation, invasion, and angiogenesis. Furthermore, the existence of multiple PCI derivatives that maintained these anticancer effects was revealed. Based on these findings, an anticancer agent containing PCI or a derivative thereof as an active ingredient has been provided.
[0025] PCIは、分子量約 57 kDaの一本鎖糖蛋白質である。本発明においては、天然 PCI 及び人工的に作製した PCIを用いることができる。ヒトおよびその他の哺乳動物の PCI が公知である（Suzuki, K. et al" J. Biol. Chem. 262:611-6, 1987)。ヒトの治療に用い る場合には、ヒト由来の PCIが好ましい。ヒト PCIのアミノ酸配列を配列番号： 2(GenBan k Accession Number: P05154)に、そして cDNAの塩基配列を配列番号： 1に示す。 本発明においては、ヒト以外の種に由来する PCIを利用することもできる。具体的に は、哺乳動物の PCIとしては、例えばマウス、ラット、またはゥシ等の PCIが公知である (Zechmeister-Machhart, M. et. al., Gene 186(1):61— 6, 1997; Wakita, T. et. al., FE BS Lett. 429(3):263-8, 1998; Yuasa, H. et. al" Thromb. Haemost. 83(2):262- 7, 200 0)。  [0025] PCI is a single-chain glycoprotein with a molecular weight of about 57 kDa. In the present invention, natural PCI and artificially produced PCI can be used. PCI of humans and other mammals is known (Suzuki, K. et al "J. Biol. Chem. 262: 611-6, 1987) When used for human therapy, human-derived PCI is The amino acid sequence of human PCI is shown in SEQ ID NO: 2 (GenBank Accession Number: P05154), and the nucleotide sequence of cDNA is shown in SEQ ID NO: 1. In the present invention, PCI derived from a species other than human is used. Specifically, as mammalian PCI, for example, PCI such as mouse, rat or ushi is known (Zechmeister-Machhart, M. et. Al., Gene 186 (1): 61— 6, 1997; Wakita, T. et. Al., FE BS Lett. 429 (3): 263-8, 1998; Yuasa, H. et. Al "Thromb. Haemost. 83 (2): 262-7 , 200 0).
[0026] 本発明において、抗癌作用を有する限り、 PCIの全長を含むポリペプチドにカロえ、 その断片配列を含むポリペプチドを用いることもできる。本発明において、抗癌作用 を維持した PCIの断片配列を含むポリペプチドは、 PCIの誘導体と言う。 PCIの全長を 含むポリペプチド、あるいはその断片配列を含むポリペプチドには、その他のぺプチ ドにより修飾された融合ポリペプチドが含まれる。以下、 PCIに加えて、その抗癌作用 を維持した誘導体を含む用語として、「PCIs」を用いる場合がある。  [0026] In the present invention, as long as it has an anticancer activity, it is possible to use a polypeptide containing the full-length PCI and a fragment sequence thereof. In the present invention, a polypeptide containing a PCI fragment sequence that maintains anticancer activity is referred to as a PCI derivative. The polypeptide containing the full length of PCI, or the polypeptide containing the fragment sequence thereof includes a fusion polypeptide modified with other peptides. Hereinafter, in addition to PCI, “PCIs” may be used as a term that includes derivatives that maintain their anticancer activity.
[0027] 本発明によって、癌細胞の転移や増殖活性は、 PCIのプロテアーゼ阻害作用には 依存しないことが示された。したがって、癌の転移および増殖の両方またはいずれか を抑制するための抗癌剤の有効成分として、プロテアーゼ阻害活性が低い、あるい は欠失した PCI断片を利用することができる。本発明において、プロテアーゼ阻害活 性が低いとは、天然の PCIと比較して、たとえば 50%以下、あるいは 30%以下、通常 10% 以下のプロテアーゼ阻害活性を言う。  [0027] According to the present invention, it has been shown that the metastasis and proliferation activity of cancer cells does not depend on the protease inhibitory action of PCI. Accordingly, a PCI fragment having low or no protease inhibitory activity can be used as an active ingredient of an anticancer agent for suppressing cancer metastasis and / or growth. In the present invention, low protease inhibitory activity refers to protease inhibitory activity of, for example, 50% or less, or 30% or less, usually 10% or less, compared to natural PCI.
[0028] プロテアーゼに対する PCI誘導体の阻害活性は、たとえば実施例に示すような方法 によって評価することができる。すなわち、活性化プロテイン C(activated protein C; A PC)を、阻害作用を評価すべき PCI誘導体とともにインキュベートする。インキュベート 後のプロテアーゼ活性を比較することによって、 PCIまたはその誘導体のプロテア一 ゼ阻害活性を評価することができる。プロテアーゼの活性は、適当な基質を与え、プ 口テアーゼによって消化される基質の量、あるいは消化の速度を指標として測定する ことができる。ペプチドに色素を結合した発色基質を用いれば、消化を光学的に追跡 することができる。実施例においては、発色基質として Glu-Pro-Arg-p-nitroanilide (S -2366)を用いた。 [0028] The inhibitory activity of a PCI derivative against a protease can be evaluated by, for example, a method as shown in the Examples. That is, activated protein C (A PC) is incubated with a PCI derivative whose inhibitory effect is to be evaluated. By comparing the protease activity after incubation, the protease inhibitory activity of PCI or its derivative can be evaluated. Protease activity can be measured by giving an appropriate substrate and using the amount of substrate digested by the protease or the rate of digestion as an indicator. Digestion can be traced optically using a chromogenic substrate with a dye attached to the peptide. In the examples, Glu-Pro-Arg-p-nitroanilide (S-2366) was used as the chromogenic substrate.
[0029] プロテアーゼ阻害活性の評価に用いる aPC(activated protein C)は、遺伝子工学的 にあるいは化学的に合成した aPCを利用することができる。 aPCは、その全長アミノ酸 配列のみならず、酵素活性を維持した断片を用いることもできる。ヒトおよびその他の 哺乳動物の aPCのアミノ酸配列は公知である（Mather, T. et al., EMBO J. 15:6822-6 831, 1996; Foster, D.C. et al., Proc. Natl. Acad. Sci. 82:4673-4677, 1985)。例えば 、市販の aPC (protein C activated, from human plasma (ヒト血漿由来)， SIGMA, #P22 00)を用いることもできる。  [0029] As aPC (activated protein C) used for the evaluation of protease inhibitory activity, aPC synthesized by genetic engineering or chemically can be used. For aPC, not only the full-length amino acid sequence but also a fragment maintaining the enzyme activity can be used. The amino acid sequences of human and other mammalian aPCs are known (Mather, T. et al., EMBO J. 15: 6822-6 831, 1996; Foster, DC et al., Proc. Natl. Acad. Sci. 82: 4673-4677, 1985). For example, commercially available aPC (protein C activated, from human plasma, SIGMA, # P2200) can also be used.
[0030] プロテアーゼ阻害活性が低い PCI誘導体として、たとえば次のようなポリペプチドを 示すことができる。これらの条件を備えたポリペプチドは、本発明における抗癌剤の 有効成分として有用である。  [0030] As a PCI derivative having low protease inhibitory activity, for example, the following polypeptides can be shown. A polypeptide having these conditions is useful as an active ingredient of an anticancer agent in the present invention.
(a) へノリン結合領域を構成するアミノ酸配列を含む PCIの断片  (a) A fragment of PCI containing the amino acid sequence that constitutes the henoline-binding region
(b) aPCによる消化部位に変異を有する変異 PCI  (b) Mutant PCI with mutation at digestion site by aPC
[0031] 続いて、本発明において有用な PCI誘導体について具体的に説明する。本発明に おけるプロテアーゼ阻害作用を欠失した PCI変異体として、たとえば、 PCIのへパリン あるいはへパリン様グリコサミノダルカンとの結合領域を含むポリペプチドを示すこと ができる。たとえばヒト PCIのアミノ酸配列中、次に示す領域は、へパリンとの結合にお いて重要な領域であるとされている (Kuhn L.A. et al, Proc Natl Acad Sci USA. 87:85 06-8510, 1990)。ヒト以外の種に由来する PCIにおいても、両者のアミノ酸配列を整列 させ、当該領域に相同な領域を同定することができる。したがって、この領域を含む P CIの断片は、本発明における PCI誘導体として利用することができる。このような断片 アミノ酸配列として、たとえば N末端側 1 354位のアミノ酸残基を含む断片を示すこ とができる。実施例において degPCIとして記載された断片（配列番号： 15)は、配列 番号： 2の 1 354位に相当するアミノ酸配列であり、プロテアーゼ阻害作用は持たな い一方で、十分な抗癌作用を示すことが確認された。 [0031] Next, PCI derivatives useful in the present invention will be specifically described. As a PCI mutant lacking the protease inhibitory action in the present invention, for example, a polypeptide containing a binding region of PCI with heparin or heparin-like glycosaminodalcan can be shown. For example, in the amino acid sequence of human PCI, the following regions are considered to be important for binding to heparin (Kuhn LA et al, Proc Natl Acad Sci USA. 87:85 06-8510, 1990). In PCI derived from species other than humans, both amino acid sequences can be aligned to identify a region homologous to the region. Therefore, a PCI fragment containing this region can be used as a PCI derivative in the present invention. As such a fragment amino acid sequence, for example, a fragment containing the amino acid residue at position 1354 on the N-terminal side is shown. You can. The fragment (SEQ ID NO: 15) described as degPCI in the Examples is an amino acid sequence corresponding to position 1354 of SEQ ID NO: 2, and has sufficient anticancer activity while having no protease inhibitory activity. It was confirmed.
1位 15位（helix A)  1st 15th (helix A)
264位— 278位 (helix H)  264th — 278th (helix H)
[0032] PCIと結合するへノ リン様ダルコサミノグルカンの例を表 1にその構造とともに示す。 [0032] Examples of heterolin-like darcosaminoglucans that bind to PCI are shown in Table 1 together with their structures.
表 1にはこれらのへパリン様ダルコサミノダルカンの生体内における分布も合わせて 示した。これらのダルコサミノダルカンに結合する PCI誘導体は、本発明に有用である  Table 1 also shows the distribution of these heparin-like dalcosaminodalcans in vivo. PCI derivatives that bind to these dalcosaminodalcans are useful in the present invention.
[表 1] [table 1]
Figure imgf000014_0001
Figure imgf000014_0001
中でもコンドロイチン硫酸は、 PCIの作用を増強する作用が大きい。あるいはデキス トラン硫酸もへパリンと同様に PCIの作用を増強することが知られている (Kazama Y. et al, Thromb Res. 48(2): 179-85, 1987 Oct 15)。したがって、コンドロイチン硫酸ゃデ キストラン硫酸との結合性を維持した PCIの変異体は、本発明にお ヽて好ま 、。  Among them, chondroitin sulfate has a large effect of enhancing the action of PCI. Alternatively, dextran sulfate is known to enhance the action of PCI as well as heparin (Kazama Y. et al, Thromb Res. 48 (2): 179-85, 1987 Oct 15). Therefore, a variant of PCI that maintains binding to chondroitin sulfate and dextran sulfate is preferred in the present invention.
[0033] PCI誘導体のへノ リンあるいはへノ リン様グリコサミノダルカンとの結合性は、任意 の方法によって評価することができる。以下、へパリンおよびへノ リン様グリコサミノグ ルカンを示す用語として、へノ^ン類を用いる。たとえば、 PCI誘導体とへパリン類との 結合を、固定化したへパリン類に対する PCI誘導体の結合を観察することによって評 価することができる。たとえばへパリンカラムを利用して、へパリンに対する PCI誘導体 の結合を比較することができる。すなわち、へパリンァガロースカラムで PCIまたは PCI 誘導体を吸着し、 NaClで溶出する。溶出分画中に含まれる PCIまたは PCI誘導体を E LISA等で定量して、カラムへの吸着レベルを比較することができる。へパリンに対す る吸着活性が大きい PCほたは PCI誘導体はより遅い分画に溶出される (Kuhn L.A. et al, Proc Natl Acad Sci USA. 87: 8506-8510, 1990)。 [0033] The binding property of PCI derivative with henolin or henolin-like glycosaminodalcan can be evaluated by any method. Hereinafter, henones are used as terms to indicate heparin and henoline-like glycosaminoglycan. For example, the binding of PCI derivatives to heparins can be assessed by observing the binding of PCI derivatives to immobilized heparins. For example, PCI derivatives for heparin using heparin columns Can be compared. In other words, PCI or PCI derivatives are adsorbed on a heparingarose column and eluted with NaCl. PCI or PCI derivatives contained in the eluted fraction can be quantified by E LISA or the like, and the adsorption level on the column can be compared. PCs and PCI derivatives with high adsorption activity to heparin are eluted in a slower fraction (Kuhn LA et al, Proc Natl Acad Sci USA. 87: 8506-8510, 1990).
[0034] 本発明における PCI誘導体として、 aPCによる消化部位に変異を有する変異 PCIを 用いることもできる。 PCIが aPCに結合して不活性ィ匕する過程で、 PCIはその C末端に 近い領域で aPCに消化されて C末端側の断片が遊離する。 aPCによって切断される 部分は 354— 355位の Arg- Serの間であることが明らかにされている (Suzuki K. et al. , J. Biol. Chem. 262(2):611-616, 1987)。そしてこの領域に変異を含む PCI誘導体は 、 aPCによって消化されず、そのプロテアーゼ阻害活性を失う。たとえば実施例に示 した PCI変異体 R354APCIは、 354位の Argを Alaに変異させた PCI誘導体である。 R35 4APCIは、 N末端領域におけるへノリンとの結合性は保持している力 aPCによる消 化部位に変異を含むために、プロテアーゼ活性を持たない。このような変異体を、本 発明における PCI誘導体として用いることもできる。  [0034] As the PCI derivative in the present invention, a mutant PCI having a mutation at the digestion site by aPC can also be used. In the process where PCI binds to aPC and inactivates, PCI is digested by aPC in the region close to its C-terminal, and the C-terminal fragment is released. The part cleaved by aPC has been shown to be between Arg-Ser at positions 354-355 (Suzuki K. et al., J. Biol. Chem. 262 (2): 611-616, 1987). ). And PCI derivatives containing mutations in this region are not digested by aPC and lose their protease inhibitory activity. For example, the PCI mutant R354APCI shown in the Examples is a PCI derivative in which Arg at position 354 is mutated to Ala. R354 4APCI does not have protease activity because it contains a mutation at the site of extinction by aPC. Such a mutant can also be used as a PCI derivative in the present invention.
[0035] 本発明における PCIは、配列番号： 2のアミノ酸配列からなるヒトポリペプチドの他、ヒ ト以外の様々な種に由来するポリペプチドであってもよい。本発明における PCI誘導 体は、抗癌作用を有する限り、 PCIのアミノ酸配列において 1またはそれ以上のァミノ 酸残基が改変された、アミノ酸配列を含むポリペプチドであってもよい。アミノ酸配列 の改変とは、アミノ酸配列における、 1つまたはそれ以上のアミノ酸の欠失、置換また は付加であってもよい。例えば、生体内における安定性、あるいは PCIの物理的およ び生物学的特性を改善するために、そのアミノ酸配列を改変することができる。ァミノ 酸配列の改変方法は、公知である。たとえば、部位特異的変異 (Kunkel T.A., Proc. Natl. Acad. Sci. USA 82: 488-92, 1985参照）、 PCR変異、カセット変異等の方法によ つてアミノ酸配列を改変したポリペプチド、ある 、はそれをコードする DNAを得ること ができる。  [0035] PCI in the present invention may be a polypeptide derived from various species other than human in addition to the human polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. The PCI derivative in the present invention may be a polypeptide containing an amino acid sequence in which one or more amino acid residues are modified in the amino acid sequence of PCI as long as it has an anticancer activity. A modification of the amino acid sequence may be a deletion, substitution or addition of one or more amino acids in the amino acid sequence. For example, the amino acid sequence can be modified to improve in vivo stability or the physical and biological properties of PCI. Methods for modifying amino acid sequences are known. For example, there are polypeptides whose amino acid sequences have been modified by methods such as site-specific mutation (see Kunkel TA, Proc. Natl. Acad. Sci. USA 82: 488-92, 1985), PCR mutation, cassette mutation, etc. Can get the DNA that encodes it.
[0036] このような変異体は、元のアミノ酸配列と少なくとも 70%、より好ましくは少なくとも 75 %、より好ましくは少なくとも 80%、さらに好ましくは少なくとも 85%、さらにより好ましく は少なくとも 90%、そして、最も好ましくは少なくとも 95%のアミノ酸配列の同一性を有 する。本明細書において配列の同一性は、配列同一性が最大となるように必要に応 じ配列を整列化し、適宜ギャップ導入した後、元となった PCIのアミノ酸配列の残基と 同一の残基の割合として定義される。 [0036] Such a variant has at least 70%, more preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, even more preferably with the original amino acid sequence. Have at least 90% and most preferably at least 95% amino acid sequence identity. In this specification, the sequence identity refers to residues that are the same as the residues of the original PCI amino acid sequence after aligning the sequences as necessary to maximize sequence identity and introducing gaps as appropriate. Is defined as the percentage of
[0037] アミノ酸残基を改変する場合には、アミノ酸側鎖の性質が保存されている別のアミノ 酸残基に置換することにより、蛋白質の性質を維持することができる。例えば次のよう な特徴を有するアミノ酸は、互いに類似した特長を持ち、相互に置換したときに、蛋 白質の活性が維持される可能性が高い。なお括弧内に記載したアルファベットは、い ずれもアミノ酸の一文字表記を表す。 [0037] When the amino acid residue is modified, the property of the protein can be maintained by substituting it with another amino acid residue in which the property of the amino acid side chain is conserved. For example, amino acids having the following characteristics have similar characteristics to each other, and the protein activity is likely to be maintained when they are substituted with each other. Note that the alphabets in parentheses all represent one letter of amino acids.
疎水性アミノ酸（A、 I、レ M、 F、 P、 W、 Y、 V)  Hydrophobic amino acids (A, I, RE M, F, P, W, Y, V)
親水性アミノ酸（R、 D、 N、 C、 E、 Q、 G、 H、 K、 S、 T)  Hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T)
脂肪族側鎖を有するアミノ酸 (G、 A、 V、 L、 I、 P)  Amino acids with aliphatic side chains (G, A, V, L, I, P)
水酸基含有側鎖を有するアミノ酸 (S、 Τ、 Υ)  Amino acids with hydroxyl-containing side chains (S, Τ, Υ)
硫黄原子含有側鎖を有するアミノ酸 (C、 M)  Amino acids with side chains containing sulfur atoms (C, M)
カルボン酸及びアミド含有側鎖を有するアミノ酸 (D、 N、 E、 Q)  Amino acids with side chains containing carboxylic acids and amides (D, N, E, Q)
塩基含有側鎖を有するアミノ離 (R、 K、 Η)  Amino groups with base-containing side chains (R, K, Η)
芳香族含有側鎖を有するアミノ酸 (H、 F、 Y、 W)  Amino acids with aromatic-containing side chains (H, F, Y, W)
[0038] これらの各グループ内のアミノ酸の置換は、保存的置換と呼ばれる。あるアミノ酸配 列に対する 1または複数個のアミノ酸残基の欠失、付加及び Z又は他のアミノ酸によ る置換により修飾されたアミノ酸配列を有するポリペプチドがその生物学的活性を維 持することはすでに知られている（Mark, D.F. et al., Proc. Natl. Acad. Sci. USA 81:5 662-6, 1984; Zoller, M.J. and Smith, M., Nucleic Acids Res. 10:6487-500, 1982; W ang, A. et al" Science 224:1431-3, 1984; Dalbadie - McFarland, G. et al., Proc. Natl . Acad .Sci. USA 79:6409-13, 1982)。本発明において変異するアミノ酸数は特に制 限されない。通常、全長アミノ酸配列を構成するアミノ酸の 40%以内であり、好ましく は 35%以内であり、さらに好ましくは 30%以内（例えば、 25%以内）である。アミノ酸配 列の同一性は、以下の方法により決定することができる。 [0038] Substitutions of amino acids within each of these groups are referred to as conservative substitutions. A polypeptide having an amino acid sequence modified by deletion, addition and substitution of one or more amino acid residues to a certain amino acid sequence and substitution by Z or other amino acids retains its biological activity. Already known (Mark, DF et al., Proc. Natl. Acad. Sci. USA 81: 5 662-6, 1984; Zoller, MJ and Smith, M., Nucleic Acids Res. 10: 6487-500, Wang, A. et al "Science 224: 1431-3, 1984; Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA 79: 6409-13, 1982). The number of amino acids to be mutated is not particularly limited, and is usually within 40%, preferably within 35%, more preferably within 30% (for example, within 25%) of the amino acids constituting the full-length amino acid sequence. The identity of amino acid sequences can be determined by the following method.
[0039] 具体的には、塩基配列ある 、はアミノ酸配列の同一性は、 Karlin and Altschulによ るアルゴリズム BLAST (Karlin S. and Altschul S.F., Proc. Natl. Acad. Sci. USA 90:58 73-7, 1993)によって決定することができる。このアルゴリズムに基づいて、 BLASTNや BLASTXと呼ばれるプログラムが開発されている（Altschul S.F. et al.， J. Mol. Biol. 2 15:403-10, 1990) o BLASTに基づいて BLASTNによって塩基配列を解析する場合に は、パラメータ一は例えば score = 100、 wordlength = 12とする。また、 BLASTに基づ いて BLASTXによってアミノ酸配列を解析する場合には、パラメータ一は例えば score = 50、 wordlength = 3とする。 BLASTと Gapped BLASTプログラムを用いる場合には、 各プログラムのデフォルトパラメーターを用いる。これらの解析方法の具体的な手法 は公知である（NCBI (National Center for Biotechnology Information)の BLAST (Ba sic Local Alignment Search Tool)のウェブサイトを参照； http://www.ncbi.nlm.nih. go v)。 [0039] Specifically, the identity of the amino acid sequence is determined by Karlin and Altschul. Can be determined by the algorithm BLAST (Karlin S. and Altschul SF, Proc. Natl. Acad. Sci. USA 90:58 73-7, 1993). Based on this algorithm, programs called BLASTN and BLASTX have been developed (Altschul SF et al., J. Mol. Biol. 2 15: 403-10, 1990). O Base sequence analysis by BLASTN based on BLAST In this case, the parameter 1 is, for example, score = 100 and wordlength = 12. When the amino acid sequence is analyzed by BLASTX based on BLAST, the parameter 1 is, for example, score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, use the default parameters of each program. Specific methods of these analysis methods are known (refer to NCBI (National Center for Biotechnology Information) BLAST (Basic Local Alignment Search Tool) website; http: //www.ncbi.nlm.nih. go v).
[0040] 本発明にお 、て、 PCIおよびその抗癌作用を維持した誘導体を用いることができる 。 PCIおよび PCI誘導体の抗癌作用は、たとえば実施例に示すような方法によって確 認することができる。具体的には、癌細胞の浸潤、増殖、転移、あるいは血管新生等 の作用に与える影響を確認するために、以下のような方法を利用することができる。こ れらの方法によって候補ポリペプチドの抗癌作用を確認し、浸潤、増殖、転移、ある いは血管新生の少なくともひとつの抗癌作用を確認できたポリペプチドを本発明に おける PCI誘導体として選択することができる。抗癌作用は、抗癌作用を有しない対 照ポリペプチドとの比較によって評価することができる。ポリペプチドをコードする DN Aを保持したベクターを使って抗癌作用を評価するときには、当該 DNAを含まな 、ベ クタ一 (Mock)を対照とすることもできる。あるいは抗癌作用を有することが確認されて いるポリペプチドを対照とすることで、同程度、あるいはそれ以上の抗癌作用を有す ることを確認することちでさる。  [0040] In the present invention, PCI and a derivative maintaining its anticancer activity can be used. The anticancer activity of PCI and PCI derivatives can be confirmed, for example, by the method as shown in the Examples. Specifically, the following methods can be used to confirm the effects on cancer cell invasion, proliferation, metastasis, or angiogenesis. Using these methods, the anticancer activity of the candidate polypeptide is confirmed, and a polypeptide that has confirmed at least one anticancer activity of invasion, proliferation, metastasis, or angiogenesis is selected as a PCI derivative in the present invention. can do. Anticancer activity can be evaluated by comparison with a reference polypeptide that does not have anticancer activity. When the anticancer activity is evaluated using a vector carrying the DNA encoding the polypeptide, the vector (Mock) without the DNA can also be used as a control. Alternatively, by using a polypeptide that has been confirmed to have anticancer activity as a control, it can be confirmed by confirming that it has the same or higher anticancer activity.
[0041] [浸？閏： in vitro浸？閏アツセィ法 (in vitro invasion assay) ] [0041] [Immersion?閏: In vitro immersion? [In vitro invasion assay]
マトリゲル中で癌細胞の浸潤を観察することができる。癌細胞を PCI誘導体の存在 下でインキュベーションすることによって、 PCI誘導体が癌細胞の浸潤に与える影響 を確認することができる。  Invasion of cancer cells can be observed in Matrigel. By incubating cancer cells in the presence of PCI derivatives, the effect of PCI derivatives on cancer cell invasion can be confirmed.
[増殖： in vivoにおける腫 ¾?増殖の評価 (evaluation of tumor growth in vivo) ] PCI誘導体をコードする DNAで形質転換された癌細胞の増殖を観察することによつ て、 PCI誘導体が癌細胞の増殖に与える影響を評価することができる。癌細胞として はたとえば乳癌細胞 MDA-231を利用することができる。癌細胞は SCIDマウスに移植 して、生体中における癌細胞の増殖に与える影響を評価することができる。 [Evaluation of tumor growth in vivo] By observing the growth of cancer cells transformed with DNA encoding a PCI derivative, the effect of the PCI derivative on the growth of cancer cells can be evaluated. As the cancer cell, for example, breast cancer cell MDA-231 can be used. Cancer cells can be transplanted into SCID mice to evaluate the effect on cancer cell growth in vivo.
[転移：実験的月巿転移のアツセィ法 (assay of experimental lung metastasis) ] 生体内における評価系を利用して、 PCI誘導体が癌細胞の転移に与える影響を評 価することもできる。たとえば、乳癌細胞 MDA-231を SCIDマウスの尾静脈に接種し、 肺への転移を観察することができる。 PCI誘導体をコードする DNAの形質転換によつ て肺への転移が抑制されれば、 PCI誘導体の転移に対する抑制作用が確認できる。 [Metastasis: assay of experimental lung metastasis] The in vivo evaluation system can be used to evaluate the effects of PCI derivatives on cancer cell metastasis. For example, breast cancer cells MDA-231 can be inoculated into the tail vein of SCID mice and observed for metastasis to the lung. If the metastasis to the lung is suppressed by transformation of DNA encoding the PCI derivative, the inhibitory effect on the metastasis of the PCI derivative can be confirmed.
[血管新生：マトリゲル移植アツセィ法（matrigel implant assay) ] [Angiogenesis: Matrigel implant assay]
癌組織における血管新生に与える PCI誘導体の影響を、 in vivoで評価することもで きる。すなわち、癌細胞を含むマトリゲルを SCIDマウスに移植し、マトリゲル中への血 液成分の進入を指標として、癌細胞の血管新生活性を評価することができる。実施 例においては、マトリゲルを消化して、マトリゲル中に含まれるヘモグロビンの含有量 を比較した。 PCI誘導体をコードする DNAで形質転換された癌細胞において、血管 新生が抑制されれば、 PCI誘導体の血管新生に対する抑制作用を確認することがで きる。  The effect of PCI derivatives on angiogenesis in cancer tissues can also be evaluated in vivo. That is, Matrigel containing cancer cells can be transplanted into SCID mice, and the angiogenic activity of cancer cells can be evaluated using the entry of blood components into Matrigel as an index. In the examples, Matrigel was digested and the content of hemoglobin contained in Matrigel was compared. If angiogenesis is inhibited in cancer cells transformed with DNA encoding a PCI derivative, the inhibitory effect of the PCI derivative on angiogenesis can be confirmed.
[血管新生： -ヮトリ漿尿膜アツセィ法（Chick chorioallantoic membrane assay: し AM— assay)」  [Angiogenesis: -Chick chorioallantoic membrane assay: AM-assay]
トリの漿尿膜上で癌細胞を培養して、漿尿膜における血管新生を観察することによ り、癌細胞の血管新生活性を評価することができる。 PCI誘導体をコードする DNAで 形質転換された癌細胞において、血管新生が抑制されれば、 PCI誘導体の血管新生 に対する抑制作用を確認することができる。  By culturing cancer cells on the chorioallantoic membrane and observing angiogenesis in the chorioallantoic membrane, the angiogenic activity of the cancer cells can be evaluated. If angiogenesis is suppressed in cancer cells transformed with DNA encoding a PCI derivative, the inhibitory action of the PCI derivative on angiogenesis can be confirmed.
[血管新生： in vitro血管新生アツセィ法 n vitro angiogenesis assay) ]  [Angiogenesis: in vitro angiogenesis assay)
マトリゲル中で癌細胞を培養するときに、マトリゲル中に癌細胞によって形成される 毛細管を指標として、癌細胞の血管新生活性を評価することができる (Schnaper H.W . et al.， J. Cell Physiol. 65:107-18, 1995)。 PCI誘導体をコードする DNAで形質転換 された癌細胞において、血管新生が抑制されれば、 PCI誘導体の血管新生に対する 抑制作用を確認することができる。 When culturing cancer cells in Matrigel, the angiogenic activity of cancer cells can be evaluated using the capillaries formed by the cancer cells in Matrigel as an index (Schnaper HW. Et al., J. Cell Physiol 65: 107-18, 1995). If angiogenesis is suppressed in cancer cells transformed with DNA encoding a PCI derivative, the PCI derivative can The inhibitory action can be confirmed.
[0043] 本発明において、癌細胞として乳癌細胞 MDA-231を用いることで、癌細胞の増殖、 浸潤、転移、および血管新生等の活性を生体内で評価することができる。すなわち、 MDA-231を SCIDマウスに移植することによって、増殖、浸潤、転移、および血管新生 という癌の悪性化機構を実験的に再現することができる。したがって本発明は、以下 の工程を有する、被験物質の抗癌作用の評価方法を提供する。  [0043] In the present invention, by using breast cancer cell MDA-231 as a cancer cell, activities such as proliferation, invasion, metastasis, and angiogenesis of the cancer cell can be evaluated in vivo. That is, by transplanting MDA-231 into SCID mice, the malignant mechanisms of cancer such as proliferation, invasion, metastasis, and angiogenesis can be experimentally reproduced. Therefore, the present invention provides a method for evaluating the anticancer activity of a test substance, which comprises the following steps.
a)乳癌細胞 MDA-231を免疫不全動物に移植する工程；  a) transplanting breast cancer cells MDA-231 into an immunodeficient animal;
b) a)の移植の前、移植と同時、または移植の後のいずれかのタイミングで、被験物 質を MDA-231に接触させる工程；  b) contacting the test substance with MDA-231 before, at the same time as, or after the transplantation in a);
c) MDA-231の癌細胞としての活性を測定する工程；および  c) measuring the activity of MDA-231 as a cancer cell; and
d) c)で測定した活性が対照と比較して低い場合に被験物質の抗癌作用が検出され る工程。  d) A step in which the anticancer activity of the test substance is detected when the activity measured in c) is low compared to the control.
本発明において、癌細胞としての活性とは、細胞の増殖、浸潤、転移、および血管 新生力もなる群力 選択することができる。これらの活性は、たとえば先に述べたよう な方法によって測定することができる。一方上記の方法に用いる MDA-231細胞は、 MDA- MB- 231としてセルバンクから入手することができる（ATCC Accession No. HT B- 26)。  In the present invention, the activity as a cancer cell can be selected as a group force including cell proliferation, invasion, metastasis, and angiogenic potential. These activities can be measured, for example, by the method as described above. On the other hand, MDA-231 cells used in the above method can be obtained from the cell bank as MDA-MB-231 (ATCC Accession No. HT B-26).
[0044] PCIは、血液、尿、精漿、滑液または PCIを発現する細胞若しくは組織等を原料とし て、その物理的性質等に基づいて天然より単離することができる。また、公知の配列 情報に基づき、化学的に合成してもよい。 PCI誘導体については、目的とするアミノ酸 配列をコードする DNAを発現させることによって、遺伝子工学的に合成することがで きる。すなわち、遺伝子組換え技術により、 PCIまたは PCI誘導体をコードする遺伝子 で宿主細胞を形質転換することができる。遺伝子は適当な発現ベクターに組み込ん で、宿主細胞に形質転換するのが好ましい。得られた形質転換細胞を培養すること により、該細胞またはその培養上清中から、 PCほたは PCI誘導体を得ることができる 。 PCIまたは PCI誘導体の組換え体は、例えば、実施例に記載したようにして調製する ことができる。  [0044] PCI can be isolated from nature using blood, urine, seminal plasma, synovial fluid, or cells or tissues that express PCI as a raw material based on its physical properties. Alternatively, it may be chemically synthesized based on known sequence information. PCI derivatives can be synthesized by genetic engineering by expressing DNA encoding the target amino acid sequence. That is, host cells can be transformed with a gene encoding PCI or a PCI derivative by gene recombination technology. The gene is preferably incorporated into an appropriate expression vector and transformed into a host cell. By culturing the obtained transformed cells, PC or PCI derivatives can be obtained from the cells or the culture supernatant thereof. A recombinant of PCI or a PCI derivative can be prepared, for example, as described in the Examples.
[0045] 遺伝子工学的に PCほたは PCI誘導体を製造する際に適当なベクターとして、ウイ ルス、コスミド、プラスミド、ノ クテリオファージ等を利用した各種ベクターを利用するこ と力できる (Molecular Cloning 2 ed., Cold Spring Harbor Press (1989); Current Pro tocols in Molecular Biology, John Wiley & Sons (1987)) 0所望の宿主細胞内に導入 された場合に PCIが発現されるよう、ベクターは適当な制御配列を含むことができる。 PCIまたは PCI誘導体をコードする遺伝子は、該制御配列に対して読み枠がずれな いようにベクターに挿入される。ここで、 PCほたは PCI誘導体をコードする遺伝子は、 選択されたベクターおよび宿主にぉ 、て発現され得るものであれば、その種類や塩 基配列は任意である。また DNAのみならず場合によっては、 RNA等も利用することが できる。 [0045] As a vector suitable for producing a PC derivative or PCI derivative by genetic engineering, Various vectors using Rus, cosmids, plasmids, and nocteriophages can be used (Molecular Cloning 2 ed., Cold Spring Harbor Press (1989); Current Pro tocols in Molecular Biology, John Wiley & Sons ( 1987)) 0 to PCI is expressed when introduced into a desired host cell, the vector may contain appropriate control sequences. A gene encoding PCI or a PCI derivative is inserted into a vector so that the reading frame does not deviate from the control sequence. Here, the gene or base sequence of the gene encoding the PC or PCI derivative is arbitrary as long as it can be expressed in the selected vector and host. In addition to DNA, RNA can also be used in some cases.
[0046] 発現に好適な「制御配列」は、宿主細胞やベクターに応じて適宜選択することがで きる。具体的には、宿主細胞として原核細胞を選択した場合には、制御配列として、 少なくともプロモーター、リボソーム結合部位及びターミネータ一がベクターに含まれ る。真核細胞の場合には、必須の制御配列は、プロモーターおよびターミネータ一で ある。さらに必要に応じ、ェンハンサー、スプライシングシグナル、転写因子、トランス ァクチべ一ター、ポリ Aシグナル及び/またはポリアデニルイ匕シグナル等を含むことが できる。  A “control sequence” suitable for expression can be appropriately selected depending on the host cell or vector. Specifically, when a prokaryotic cell is selected as the host cell, the vector contains at least a promoter, a ribosome binding site and a terminator as control sequences. In the case of eukaryotic cells, the essential regulatory sequence is a promoter and a terminator. Furthermore, an enhancer, a splicing signal, a transcription factor, a transactivator, a poly A signal and / or a polyadenylation signal can be included as necessary.
[0047] PCほたは PCI誘導体を発現するためのベクターは、さらに必要に応じ、選択可能な マーカーを含んで 、てもよ 、。マーカーによって形質転換された宿主細胞を容易に 選択することができる。さらに、細胞内で発現された PCIまたは PCI誘導体を、小胞体 内腔若しくは細胞外、または、宿主がグラム陰性菌の場合にはペリブラズム内への移 行させるためにシグナルペプチドコード配列を PCI遺伝子または PCI誘導体遺伝子に 付加するようにしてベクターに組み込んでもよい。このようなシグナルペプチドは、選 択された宿主細胞において正確に認識されればよぐ PCI固有のものでも、異種蛋白 質由来のものであってもよい。また、必要に応じリンカ一、開始コドン及び終止コドン 等を付加してもよい。たとえば配列番号： 2に記載されたアミノ酸配列 406アミノ酸残 基中、 N末端の 1 19位はヒト PCI固有のシグナル配列である。したがって、この領域 のアミノ酸配列を他のシグナル配列に組み換えることもできる。  [0047] A vector for expressing a PC or PCI derivative may further contain a selectable marker, if necessary. Host cells transformed with the marker can be easily selected. In addition, the signal peptide coding sequence can be transferred to the PCI gene or PCI derivative to transduce intracellularly expressed PCI or PCI derivatives into the endoplasmic reticulum lumen or extracellular, or into periplasm if the host is a Gram-negative bacterium. It may be incorporated into a vector so as to be added to a PCI derivative gene. Such a signal peptide may be unique to PCI, as long as it is recognized correctly in the selected host cell, or may be derived from a heterologous protein. If necessary, a linker, a start codon, a stop codon, etc. may be added. For example, among the 406 amino acid residues described in SEQ ID NO: 2, the N-terminal position 119 is a signal sequence unique to human PCI. Therefore, the amino acid sequence in this region can be recombined with other signal sequences.
[0048] 遺伝子は、制限酵素部位を利用したリガーゼ反応により、ベクターに挿入すること 力 Sできる (Molecular Cloning 2n ed., Cold Spring Harbor Press (1989)Section5.61— 5. 63; Current Protocols in Molecular Biology, John Wiley & Sons (1987)11.4-11.11)。 また、使用する宿主細胞のコドン使用頻度を考慮して、高い発現効率が得られる塩 基配列を選択し、ベクターを設計することができる（Grantham R. et al.， Nucleic Acids Res. 9:r43-74, 1981)。 [0048] The gene may be inserted into the vector by a ligase reaction using a restriction enzyme site. Can force S (Molecular Cloning 2 n ed, Cold Spring Harbor Press (1989) Section5.61- 5. 63;. Current Protocols in Molecular Biology, John Wiley & Sons (1987) 11.4-11.11). In addition, considering the codon usage of the host cell to be used, a vector can be designed by selecting a base sequence that provides high expression efficiency (Grantham R. et al., Nucleic Acids Res. 9: r43). -74, 1981).
[0049] 適当な宿主に導入して PCIまたは PCI誘導体を作製する場合には、上述の発現べク ターと適当な宿主との組み合わせを使用することができる。真核細胞を宿主として使 用する場合、動物細胞、植物細胞及び真菌細胞を用いることができる。宿主として利 用できる細胞を以下に例示する。  [0049] When PCI or a PCI derivative is produced by introduction into an appropriate host, a combination of the above expression vector and an appropriate host can be used. When eukaryotic cells are used as hosts, animal cells, plant cells, and fungal cells can be used. Examples of cells that can be used as hosts are shown below.
[動物細胞]  [Animal cells]
(1)哺乳類細胞：例えば、 CHO, COS,ミエローマ、 BHK (baby hamster kidney) , He La, Vero,  (1) Mammalian cells: For example, CHO, COS, myeloma, BHK (baby hamster kidney), He La, Vero,
(2)両生類細胞:例えば、アフリカッメガエル卵母細胞  (2) Amphibian cells: Xenopus oocytes, for example
(3)昆虫細胞:例えば、 S19, S121, Tn5等、若しくはカイコ等の個体  (3) Insect cells: For example, S19, S121, Tn5, etc., or individuals such as silkworms
[植物細胞]  [Plant cells]
ニコティアナ (Nicotiana)属：例えばニコティアナ'タノくカム (Nicotiana tabacum)由来 の細胞をカルス培養する  Nicotiana genus: for example, calli culture of cells from Nicotiana tabacum
[真菌細胞]  [Fungal cells]
酵母：例えば、サッカロミセス (Saccharomyces)属；例えばサッカロミセス ·セレピシェ（ ¾accharomyces serevisiae)  Yeast: for example, Saccharomyces genus; eg Saccharomyces serevisiae
糸状菌：例えば、ァスペルギルス (Aspergillus)属；例えばアスペスギルス ·二ガー（As pergillus niger等  Filamentous fungi: for example, Aspergillus genus; for example Aspergillus niger
[原核細胞]  [Prokaryotic cells]
細菌細胞：例えば大腸菌（Escherichia coli)、枯草菌（Bacillus subtilis)  Bacterial cells: for example, Escherichia coli, Bacillus subtilis
これらの細胞に、 PCI遺伝子または PCI誘導体遺伝子を形質転換により導入し、形 質転換された細胞を in vitroで培養することにより PCほたは PCI誘導体が得られる。  PC or PCI derivatives can be obtained by introducing the PCI gene or PCI derivative gene into these cells by transformation and culturing the transformed cells in vitro.
[0050] 宿主細胞の形質転換は、選択した宿主及びベクターに適した方法を採用すればよ い。例えば、原核細胞を宿主とする場合には、カルシウム処理及びエレクトポレーショ ン等による方法が知られている。植物細胞についてはァグロバタテリゥム法を、哺乳 動物細胞についてはリン酸カルシウム沈降法を例示できる。本発明は、特にこれらの 方法に限定されるわけではなぐ公知の核マイクロインジェクション、細胞融合、電気 パルス穿孔法、プロトプラスト融合、リポフエクタミン法（GIBCO BRL)、 DEAE-デキスト ラン法、 FuGENE6試薬 (Boehringer- Mannheim)を用いた方法を包含する様々な方法 を採用することができる。 [0050] For transformation of the host cell, a method suitable for the selected host and vector may be employed. For example, when prokaryotic cells are used as hosts, calcium treatment and electroporation The method by the method etc. is known. For plant cells, the agrobacterium method can be used, and for mammalian cells, the calcium phosphate precipitation method can be used. The present invention is not particularly limited to these methods, but known nuclear microinjection, cell fusion, electric pulse perforation, protoplast fusion, lipophectamine method (GIBCO BRL), DEAE-dextran method, FuGENE6 reagent (Boehringer- Various methods including the method using Mannheim) can be adopted.
[0051] 宿主細胞の培養は、選択した細胞に適した公知の方法にしたがって行うことができ る。例えば、動物細胞を宿主とする場合、 DMEM、 MEM, RPMI_1640、 199または IMD M等の培地を用い、必要に応じゥシ胎児血清 (FCS)等を添加して、 pH約 6〜8、 30〜 40°Cにおいて 15〜200時間前後の培養を行うことができる。その他、培養中、必要に 応じ培地の交換、通気、攪拌等の必要とされる操作を行うことができる。 [0051] Host cell culture can be performed according to a known method suitable for the selected cell. For example, when animal cells are used as a host, a medium such as DMEM, MEM, RPMI_1640, 199 or IMD M is used, and urine fetal serum (FCS) is added as necessary, so that pH is about 6 to 8, 30 to Cultivation can be performed at 40 ° C for about 15 to 200 hours. In addition, necessary operations such as medium exchange, aeration, and agitation can be performed as needed during culture.
[0052] PCIおよび PCI誘導体は、公知の手法により精製して用いることが好ま 、。 PCIまた は PCI誘導体は、一般的な蛋白質の精製方法に従って均一に精製することができる 。例えば、次に示すような精製技術を適宜選択し、組み合わせて目的とする蛋白質 を分離 製すること力 Sでさる (Strategies for Protein Purification and Charcterization: A Laboratoy Course Manual, Daniel R.Marshak et al. eds., Cold Spring Harbor Lab oratory Press (1996) ; Antibodies： A Laboratory Manual. Ed Harlow and David Lane , Cold Spring Harbor Laboratory(1988))。 [0052] PCI and PCI derivatives are preferably purified and used by a known method. PCI or PCI derivatives can be purified uniformly according to general protein purification methods. For example, the following purification techniques can be selected as appropriate and combined to separate and produce the target protein with S (Strategies for Protein Purification and Charcterization: A Laboratoy Course Manual, Daniel R. Marshak et al. Eds , Cold Spring Harbor Laboratory Press (1996); Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)).
クロマトグラフィーカラム  Chromatography column
フイノレター  Huino Letter
限外濾過  Ultrafiltration
塩析  Salting out
透析  Dialysis
調製用ポリアクリルアミドゲル電気泳動  Preparative polyacrylamide gel electrophoresis
等電点電気泳動等  Isoelectric focusing etc.
本発明における精製方法は、これらに限定されない。また本発明に利用できるクロ マトグラフィ一としては、次のようなクロマトグラフィーを示すことができる。  The purification method in the present invention is not limited to these. Further, as a chromatograph that can be used in the present invention, the following chromatography can be shown.
ァフィ二ティークロマトグラフィー イオン交換クロマトグラフィー Affinity chromatography Ion exchange chromatography
疎水性クロマトグラフィー  Hydrophobic chromatography
ゲル濾過  Gel filtration
逆相クロマトグラフィー  Reversed phase chromatography
吸着クロマトグラフィー等  Adsorption chromatography etc.
これらのクロマトグラフィーは、 HPLCや FPLC等の液相クロマトグラフィーを用いて行 うことができる。ァフィユティークロマトグラフィーには、例えば、 PCIに対する抗体を用 いることがでさる。  These chromatographies can be performed using liquid phase chromatography such as HPLC and FPLC. For example, an antibody against PCI can be used in the affinity chromatography.
[0053] 本発明の抗癌剤は、上述のようにして得られる PCほたは PCI誘導体を有効成分とし て含有する。有効成分として配合される PCIまたは PCI誘導体は単一であってもよ ヽし 、複数種の PCIおよび Zまたは PCI誘導体を配合することもできる。なお、 PCIおよび Zまたは PCI誘導体を「有効成分として含有する」とは、 PCIおよび Zまたは PCI誘導 体を活性成分の少なくとも 1つとして含むことを言う。抗癌剤における有効成分の含 有率は限定されない。また、本発明の抗癌剤は、 PCIおよび Zまたは PCI誘導体と合 わせて、抗癌作用を有する他の有効成分を含有することができる。  [0053] The anticancer agent of the present invention contains a PC or PCI derivative obtained as described above as an active ingredient. A single PCI or PCI derivative may be blended as an active ingredient, or multiple types of PCI and Z or PCI derivatives may be blended. “Containing PCI and Z or a PCI derivative as an active ingredient” means containing PCI and Z or a PCI derivative as at least one active ingredient. The content of the active ingredient in the anticancer agent is not limited. In addition, the anticancer agent of the present invention can contain other active ingredients having anticancer activity in combination with PCI and Z or PCI derivatives.
[0054] PCIおよび PCI誘導体は、常法に従って製剤化することができる（例えば、 Remingto n s Pharmaceutical Science, latest edition, Mark Publishing company, Easton, U.¾. A)。さらに、必要に応じ、医薬的に許容される担体及び/または添加物を供に含むこ ともできる。例えば、界面活性剤（PEG、 Tween等）、賦形剤、酸化防止剤（ァスコルビ ン酸等)、着色料、着香料、保存料、安定剤、緩衝剤 (リン酸、クェン酸、他の有機酸 等)、キレート剤 (EDTA等)、懸濁剤、等張化剤、結合剤、崩壊剤、滑沢剤、流動性 促進剤、矯味剤等を含むことができる。し力しながら、本発明の抗癌剤は、その他常 用の担体を含むこともできる。  [0054] PCI and PCI derivatives can be formulated according to a conventional method (for example, Remington Pharmaceutical Science, latest edition, Mark Publishing company, Easton, U.¾. A). Furthermore, a pharmaceutically acceptable carrier and / or additive can be included as required. For example, surfactants (PEG, Tween, etc.), excipients, antioxidants (ascorbic acid, etc.), coloring agents, flavoring agents, preservatives, stabilizers, buffering agents (phosphoric acid, citrate, other organics) Acid), chelating agents (EDTA, etc.), suspending agents, tonicity agents, binders, disintegrating agents, lubricants, fluidity promoters, flavoring agents, and the like. However, the anticancer agent of the present invention can also contain other conventional carriers.
具体的には、軽質無水ケィ酸、乳糖、結晶セルロース、マン-トール、デンプン、力 ノレメロースカルシウム、カノレメロースナトリウム、ヒドロキシプロピノレセルロース、ヒドロキ シプロピルメチルセルロース、ポリビュルァセタールジェチルァミノアセテート、ポリビ -ルピロリドン、ゼラチン、中鎖脂肪酸トリダリセライド、ポリオキシエチレン硬化ヒマシ 油 60、白糖、カルボキシメチルセルロース、コーンスターチ、無機塩類等を担体として 挙げることができる。また、その他の低分子量のポリペプチド、血清アルブミン、ゼラ チン及び免疫グロブリン等の蛋白質、並びに、グリシン、グルタミン、ァスパラギン、ァ ルギニン及びリシン等のアミノ酸を含むこともできる。 Specifically, light anhydrous carboxylic acid, lactose, crystalline cellulose, mannitol, starch, strength nomellose calcium, canolemellose sodium, hydroxypropinolecellulose, hydroxypropylmethylcellulose, polybulacetal jetylamino Acetate, polyvinylpyrrolidone, gelatin, medium-chain fatty acid tridalylide, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, inorganic salts, etc. as carriers Can be mentioned. It may also contain other low molecular weight polypeptides, proteins such as serum albumin, gelatin and immunoglobulin, and amino acids such as glycine, glutamine, asparagine, arginine and lysine.
注射用の水溶液とする場合には、 PCIおよび Zまたは PCI誘導体を、例えば、生理 食塩水、ブドウ糖またはその他の補助薬を含む等張液に溶解する。補助薬としては、 例えば、 D-ソルビトール、 D-マンノース、 D-マン-トール、塩化ナトリウムが挙げられ 、さらに、適当な溶解補助剤、例えばアルコール (エタノール等）、ポリアルコール (プ ロピレンダリコール、 PEG等）、非イオン性界面活性剤（ポリソルベート 80、 HCO-50) 等と併用してもよい。  In the case of an aqueous solution for injection, PCI and Z or PCI derivatives are dissolved in an isotonic solution containing, for example, physiological saline, glucose or other adjuvants. Adjuvants include, for example, D-sorbitol, D-mannose, D-mannitol, sodium chloride, and further suitable solubilizers such as alcohol (ethanol, etc.), polyalcohol (propylene alcohol, PEG, etc.), nonionic surfactants (polysorbate 80, HCO-50), etc. may be used in combination.
[0055] また、必要に応じ PCIおよび Zまたは PCI誘導体をマイクロカプセル (ヒドロキシメチ ルセルロース、ゼラチン、ポリ [メチルメタクリル酸]等のマイクロカプセル）に封入したり 、コロイドドラッグデリバリーシステム (リボソーム、アルブミンミクロスフエア、マイクロエ マルジヨン、ナノ粒子及びナノカプセル等)とすることもできる（"Remington's Pharmace utical Science 16th edition", Oslo Ed. (1980)等参照）。さらに、薬剤を徐放性の薬剤 とする方法も公知である（Langer et al.， J. Biomed. Mater. Res. 15: 167-277, 1981; L anger, Chem. Tech. 12:98-105, 1982;米国特許第 3, 773,919号;欧州特許出願公開（ EP)第 58,481号； Sidman K.R. et al, Biopolymers 22:547-56, 1983; EP第 133,988号） 。これらの公知の製剤技術を本発明に応用することができる。 [0055] If necessary, PCI and Z or PCI derivatives can be encapsulated in microcapsules (microcapsules such as hydroxymethylcellulose, gelatin, poly [methylmethacrylic acid]) or colloid drug delivery systems (ribosomes, albumin micross). Fuea, Maikuroe Marujiyon may be a nano-particles, and nano-capsules) ( "Remington's Pharmace utical Science 16 th edition", Oslo Ed. (1980) see the like). Furthermore, a method of making a drug a sustained-release drug is also known (Langer et al., J. Biomed. Mater. Res. 15: 167-277, 1981; Langer, Chem. Tech. 12: 98-105). 1982; U.S. Pat. No. 3,773,919; European Patent Application Publication (EP) 58,481; Sidman KR et al, Biopolymers 22: 547-56, 1983; EP 133,988). These known formulation techniques can be applied to the present invention.
[0056] 本発明の抗癌剤は、経口または非経口のいずれの経路によっても投与することが できる。好ましい投与方法は、非経口投与である。具体的には、注射、経鼻投与、経 肺投与及び経皮投与により患者に投与される。注射剤型の例としては、例えば、静 脈内注射、筋肉内注射、腹腔内注射または皮下注射等により全身または局部的に 投与することができる。  [0056] The anticancer agent of the present invention can be administered by either oral or parenteral routes. A preferred method of administration is parenteral administration. Specifically, it is administered to patients by injection, nasal administration, pulmonary administration, and transdermal administration. Examples of the injection form can be administered systemically or locally by, for example, intravenous injection, intramuscular injection, intraperitoneal injection or subcutaneous injection.
また、患者の年齢、症状により適宜投与方法を選択することができる。投与量として は、例えば、一回につき体重 1 kgあたり 0.0001 mgから 1000 mgの範囲で選ぶことが可 能である。または、例えば、患者あたり 0.001〜100000 mg/bodyの範囲で投与量を選 ぶことができる。し力しながら、本発明の抗癌の投与量は、これらの投与量に制限さ れない。 [0057] また、上述の PCIおよび Zまたは PCI誘導体をコードする遺伝子を遺伝子治療用べ クタ一に組込み、遺伝子治療を行うための抗癌剤とすることもできる。遺伝子の投与 方法としては、 nakedプラスミドによる直接投与の他、リボソーム等にパッケージングす ることもできる。あるいは各種の遺伝子治療用ベクターを利用することもできる。たとえ ば以下のような遺伝子治療用の各種ウィルスベクターが公知である (Adolph K.W. ed ., Viral Genome Methods, CRC Press, Florid (1996)参照）。 The administration method can be appropriately selected depending on the age and symptoms of the patient. The dose can be selected, for example, in the range of 0.0001 mg to 1000 mg per kg body weight. Alternatively, for example, the dose can be selected in the range of 0.001 to 100,000 mg / body per patient. However, the dose of the anti-cancer of the present invention is not limited to these doses. [0057] In addition, a gene encoding the above-described PCI and Z or PCI derivative can be incorporated into a vector therapy vector to provide an anticancer drug for gene therapy. As a gene administration method, in addition to direct administration using a naked plasmid, it can be packaged in a ribosome or the like. Alternatively, various gene therapy vectors can be used. For example, the following various viral vectors for gene therapy are known (see Adolph KW ed., Viral Genome Methods, CRC Press, Florid (1996)).
レトロウイノレスベタター  Retro Winores Better
アデノウイルスベクター  Adenovirus vector
ワクシニアウィルスベクター  Vaccinia virus vector
ボックスウイノレスベタター  Box Winores Better
アデノ随伴ウィルスベクター  Adeno-associated virus vector
而ベクター等  Meta vector etc.
[0058] あるいは、コロイド金粒子等のビーズ担体に遺伝子を被覆 (WO93/17706等)して投 与することができる。生体内において PCIまたは PCI誘導体が発現され、その作用を 発揮できる限り、遺伝子の投与方法は限定されない。好ましくは、適当な非経口経路 を介して遺伝子を注射、あるいは注入することにより十分な量が投与される。非経口 経路としては、静脈内、腹腔内、皮下、皮内、脂肪組織内、乳腺組織内、吸入若しく は筋肉内の経路、ガス誘導性粒子衝撃法 (電子銃等による）、添鼻薬等粘膜経路を 介する方法等を示すことができる。 ex vivoにおいて遺伝子を細胞に導入し、該細胞 を動物に戻すことにより PCほたは PCI誘導体をコードする遺伝子を投与することもで きる。 ex vivoにおける遺伝子の導入には、リボソームトランスフエクシヨン、粒子衝撃 法 (米国特許第 4,945,050号)、またはウィルス感染等を利用することができる。  [0058] Alternatively, the gene can be coated on a bead carrier such as colloidal gold particles (WO93 / 17706 or the like). The gene administration method is not limited as long as PCI or a PCI derivative is expressed in vivo and can exert its action. Preferably, a sufficient amount is administered by injection or infusion of the gene via a suitable parenteral route. Parenteral routes include intravenous, intraperitoneal, subcutaneous, intradermal, adipose tissue, mammary tissue, inhalation or intramuscular route, gas-induced particle bombardment method (using an electron gun, etc.), nasal medication, etc. The method through the mucosal route can be shown. A gene encoding a PC or PCI derivative can also be administered by introducing a gene into a cell ex vivo and returning the cell to an animal. For ex vivo gene introduction, ribosome transformation, particle bombardment (US Pat. No. 4,945,050), viral infection, or the like can be used.
なお本明細書において引用された全ての先行技術文献は、参照として本明細書に 組み入れられる。  All prior art documents cited in the present specification are incorporated herein by reference.
実施例  Example
[0059] 〈材料および方法〉 <Materials and methods>
1)材料  1) Material
制限エンドヌクレアーゼは、 TOYOBO、 日本、大阪力 購入した。 Taq DNAポリメラ ーゼは Roche Biochemicals (スイス、バーゼル）製、クレノー断片、 T4ポリヌクレオチド キナーゼ、および T4 DNAリガーゼは日本ジーン（日本、東京）製であった。 QuikCha nge XL部位特異的突然変異誘発キットは、 Stratagene、米国、テキサス州、シーダ一 クリークから購入した。ダイターミネータ一サイクルシークェンスレディーリアクションキ ットは、 ABI、カリフォルニア州、フォスターシティ一力 購入した。放射性ヌクレオチド ([ a - 32P]dCTPおよび [ γ - 32Ρ]ΑΤΡ)は、 Amersham Bioscience (スウェーデン、ゥプサ ラ）製であった。トランスフエクシヨン試薬 Effectene、は、 QIAGEN, Inc、 日本、東京か ら購入した。使用した他の化学薬品および試薬はすべて、市販の最等級のものであ つた o Restriction endonuclease was purchased from TOYOBO, Japan, Osaka. Taq DNA Polymer The cases were from Roche Biochemicals (Basel, Switzerland), and the Klenow fragment, T4 polynucleotide kinase, and T4 DNA ligase were from Nippon Gene (Tokyo, Japan). QuikChange XL site-directed mutagenesis kit was purchased from Stratagene, Cedar Creek, Texas, USA. The Die Terminator One-Cycle Sequence Lady Reaction Kit was purchased by ABI, Foster City, California. Radionucleotide ([a - 32 P] dCTP and [γ - 32 Ρ] ΑΤΡ) were from Amersham Bioscience (Sweden, Upusa la). Transfusion reagent Effectene was purchased from QIAGEN, Inc., Tokyo, Japan. All other chemicals and reagents used were of the highest quality on the market o
[0060] 2)細胞培養  [0060] 2) Cell culture
乳癌細胞 MDA-231は、 日本癌研究リサーチリソースバンク力 入手した。 MDA-231 は、 10%ゥシ胎仔血清 (FBSKEQUITECH- BIO、テキサス州、ケルビル）、 100 μ g/ml ペニシリン、および 100 IU/mlストレプトマイシン（三光純薬、 日本、東京）を添カ卩した ダルベッコ変法イーグル培地 (DMEM) (日水製薬、 日本、東京）中、 5% COを含むカロ  Breast cancer cell MDA-231 was obtained from Japan Cancer Research Resource Bank. MDA-231 is Dulbecco supplemented with 10% urine fetal serum (FBSKEQUITECH-BIO, Kelville, TX), 100 μg / ml penicillin, and 100 IU / ml streptomycin (Sanko Junyaku, Tokyo, Japan). Modified Eagle Medium (DMEM) (Nissui Pharmaceutical, Tokyo, Japan) with 5% CO
2 湿雰囲気中で 37°Cで培養した。  2 Incubate at 37 ° C in a humid atmosphere.
[0061] 3)原型 PCI、 R354APCI、 degPCI発現 MDA-231細胞の調製 [0061] 3) Preparation of prototype PCI, R354APCI, degPCI expressing MDA-231 cells
ヒト原型 PCI(intact PCI), R354APCI、または degPCIの発現ベクターは、以下のよう に構築した。シグナルペプチドを有する R354APCI cDNAは、次の塩基配列からなる 合成オリゴヌクレオチドプライマーを用いて、全長 PCI cDNA (Suzuki K. et al.， J. Biol . Chem. 262:611-6, 1987)を铸型として QuickChange部位特異的突然変異誘発キット (Stratagene,カリフォルニア州、ラ 'ホーャ）により調製した。下線を引いたヌクレオチ ドは変異部位を示す。  Human prototype PCI (intact PCI), R354APCI, or degPCI expression vectors were constructed as follows. R354APCI cDNA with a signal peptide is a full-length PCI cDNA (Suzuki K. et al., J. Biol. Chem. 262: 611-6, 1987) using a synthetic oligonucleotide primer consisting of the following nucleotide sequences: As prepared with the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, Calif.). Underlined nucleotides indicate mutation sites.
5し TTCACTTTCGCGTCGGCCCGC- 3' (配列番号： 3)および  5 TTCACTTTCGCGTCGGCCCGC-3 '(SEQ ID NO: 3) and
5 -GCGGGCCGACGCGAAAGTGAA-3' (配列番頭： 4)  5 -GCGGGCCGACGCGAAAGTGAA-3 '(SEQ ID NO: 4)
[0062] シグナルペプチド配列を有する degPCI cDNAは、以下のプライマー対を用いて、ポ リメラーゼ連鎖反応 (PCRXSaiki R.K. et al.， Science 239:487-91 , 1988)により調製し た。配列番号： 5と配列番号： 6で示したプライマーは、それぞれ PCIの cDNA (配列番 号： 1)の 31〜52位、ぉょび1194〜1176位に対応してぃる0¾ &31^ T. et al.， Int. J . Hematol. 58:213-224, 1993)。下線を引いたヌクレオチドは、 2つのプライマーの 5' 末端に挿入された EcoR I部位を示す。 [0062] A degPCI cDNA having a signal peptide sequence was prepared by polymerase chain reaction (PCRXSaiki RK et al., Science 239: 487-91, 1988) using the following primer pairs. The primers represented by SEQ ID NO: 5 and SEQ ID NO: 6 correspond to positions 31 to 52 and positions 1194 to 1176 of PCI cDNA (SEQ ID NO: 1), respectively. et al., Int. J Hematol. 58: 213-224, 1993). Underlined nucleotides indicate the EcoRI site inserted at the 5 'end of the two primers.
5し GCGAATTCCTCTGGCAGAGCCTCCGTTTCC- 3' (配列番号： 5)および 5 -GCGAATTCTCACCTGAAAGTGAAGATTGTCC-3 ' (配列番号： 6) [0063] 製造業者の説明に従って、 R354APCIまたは degPCIの DNA断片を pCRII-TOPOに サブクロー-ングし、大腸菌 Topi OF'に形質転換した。 ABI 310ジェネティックアナライ ザ一を用いて、 DNA配列を確認した。次いで、原型ヒト PCI cDNA (Suzuki K. et al.， J . Biol. Chem. 262:611-6, 1987)、変異体 R354APCI cDNA、または degPCI cDNAを哺 乳動物発現ベクター pRC/CMV(Invitrogen Corp.,カリフォルニア州、カールズバッド )に挿入し、 Effectanceトランスフエクシヨン試薬を用いて MDA-231にトランスフエクショ ンした。  GCGAATTCCTCTGGCAGAGCCTCCGTTTCC-3 '(SEQ ID NO: 5) and 5 -GCGAATTCTCACCTGAAAGTGAAGATTGTCC-3' (SEQ ID NO: 6) [0063] Subclone the DNA fragment of R354APCI or degPCI into pCRII-TOPO according to the manufacturer's instructions, E. coli Topi OF 'was transformed. The DNA sequence was confirmed using an ABI 310 genetic analyzer. Next, the prototype human PCI cDNA (Suzuki K. et al., J. Biol. Chem. 262: 611-6, 1987), mutant R354APCI cDNA, or degPCI cDNA was transformed into the mammalian expression vector pRC / CMV (Invitrogen Corp. , Carlsbad, Calif.) And transfected into MDA-231 using Effectance transfection reagent.
トランスフエクシヨンした後、 800 μ g/mlジエネティシンを含む DMEMを用いて、原型 P CI、 R354APCU degPCI発現 MDA-231細胞株を選択した。その後、以下に示すように 酵素免疫測定法 (ELISA)により、およびノーザンブロット解析による PCI mRNAの評価 により、各細胞株の培地中の PCI抗原を測定し、クローユングした MDA-231細胞株の 原型 PCI、 R354APCI、または degPCI発現を確認した。トランスフエクシヨンした 480の 細胞株から、大量の PCIを発現する細胞株として MDA-PCI 1および MDA-PCI 2、大 量の R354APCIを発現する細胞株として MDA- R354APCI 1および MDA- R354APCI 2 、ならびに大量の degPCIを発現する細胞株として MDA-degPCI 1および MDA-degPC I 2を選択し、実験に使用した。同様の手順に従い、 DNA挿入物を含まない pRC/CM Vをトランスフエクシヨンした MDA231細胞を調製して陰性対照として使用し、これらを MDA-Mock 1および MDA— Mock 2と命名した。  After transfection, a prototype PCI, R354APCU degPCI expressing MDA-231 cell line was selected using DMEM containing 800 μg / ml dieticin. Then, as shown below, the PCI antigen in the medium of each cell line was measured by enzyme immunoassay (ELISA) and by PCI mRNA evaluation by Northern blot analysis, and the prototype PCI of the cloned MDA-231 cell line , R354APCI, or degPCI expression was confirmed. From 480 cell lines transfected, MDA-PCI 1 and MDA-PCI 2 as cell lines expressing large amounts of PCI, MDA-R354APCI 1 and MDA-R354APCI 2 as cell lines expressing large amounts of R354APCI, and MDA-degPCI 1 and MDA-degPC I 2 were selected as cell lines expressing a large amount of degPCI and used in the experiment. Following a similar procedure, MDA231 cells transfected with pRC / CMV without DNA insert were prepared and used as negative controls and were designated MDA-Mock 1 and MDA-Mock 2.
[0064] 4)酵素免疫測定法 (ELISA)  [0064] 4) Enzyme immunoassay (ELISA)
培地中の PCIおよび uPA抗原レベルは、以前に記載したように (Wakita T. et al.， Int . J. Cancer 108:516-23, 2004)、ポリクローナル抗 PCI抗体および抗 uPA抗体を用い た酵素免疫測定法 (ELISA)により決定した。  The levels of PCI and uPA antigen in the medium were determined as described previously (Wakita T. et al., Int. J. Cancer 108: 516-23, 2004), using enzymes with polyclonal anti-PCI and anti-uPA antibodies. Determined by immunoassay (ELISA).
[0065] 5) RNA抽出  [0065] 5) RNA extraction
RNAzol B試薬（TEL-TEST、テキサス州、フレンズウッド）を用いて、改変チォシアン 酸グァ-ジン-フエノールクロ口ホルム技法により、 MDA-231, MDA-PCU MDA-R354 APCI、 MDA-degPCI、および MDA-Mock細胞から全 RNAを調製した (Chomczynski P . et al., Anal Biochem 162:156-59, 1987)。分光測定により全 RNAを定量し、使用す るまで- 80°Cで保存した。 Modified thiocyan using RNAzol B reagent (TEL-TEST, Friendswood, TX) Total RNA was prepared from MDA-231, MDA-PCU, MDA-R354 APCI, MDA-degPCI, and MDA-Mock cells using the acid guanidine-phenol closed mouth technique (Chomczynski P. et al., Anal Biochem 162 : 156-59, 1987). Total RNA was quantified spectrophotometrically and stored at -80 ° C until use.
[0066] 6)ノーザンブロット解析法 [0066] 6) Northern blot analysis
MDA- 231、 MDA-PCI, MDA- R354APCI、 MDA- degPCI、および MDA- Mock細胞に 由来する全 RNA (20 μ g)をホルムアルデヒド-ァガロースゲル中で電気泳動し、次い で GeneScreenナイロン膜（NEN Life Science,マサチューセッツ州、ボストン）に転写し た。 UV架橋した後、膜をランダムプライム32 P標識全長ヒト PCI cDNAプローブ (Suzuki K. et al., J. Biol. Chem. 262:611-6, 1987)とハイブリダィズさせた。 Total RNA (20 μg) from MDA-231, MDA-PCI, MDA-R354APCI, MDA-degPCI, and MDA-Mock cells was electrophoresed in formaldehyde-agarose gel, and then GeneScreen nylon membrane (NEN Life Science, Boston, Massachusetts). After UV crosslinking, the membrane was hybridized with a random prime 32 P-labeled full-length human PCI cDNA probe (Suzuki K. et al., J. Biol. Chem. 262: 611-6, 1987).
2x塩化ナトリウム-コハク酸ナトリウム（SSC)[300 mM NaCl、 30 mMコハク酸ナトリウム 、 pH 7.0]および 0.1%ドデシル硫酸ナトリウム (SDS)を含む溶液で、次いで lx SSCおよ び 0.1% SDSを含む溶液で、最終的に O.lx SSCおよび 0.1% SDSを含む溶液でそれぞ れ 20分間、連続して洗浄した後、膜をイメージングプレートに露光し、 BAS-2000ィメ ージアナライザー（富士写真フィルム、 日本、東京）を用いて可視化した。ヒト PCI cDN Aプローブとハイブリダィズさせた後、膜を 0.5% SDS中で煮沸し、上記のように、 32P標 識ヒトグリセルアルデヒド三リン酸脱水素酵素 (GAPDH) cDNAプローブ（Clontech、力 リフォルニァ州、パロアルト）とハイブリダィズさせた。 2x sodium chloride-sodium succinate (SSC) [300 mM NaCl, 30 mM sodium succinate, pH 7.0] and a solution containing 0.1% sodium dodecyl sulfate (SDS), followed by a solution containing lx SSC and 0.1% SDS Finally, after 20 minutes of continuous washing with a solution containing O.lx SSC and 0.1% SDS, the membrane was exposed to an imaging plate, and BAS-2000 Image Analyzer (Fuji Photo Film, Japan) , Tokyo). After hybridizing with the human PCI cDN A probe, the membrane was boiled in 0.5% SDS and the 32 P-labeled human glyceraldehyde triphosphate dehydrogenase (GAPDH) cDNA probe (Clontech, Power Refolder) as described above. Palo Alto).
[0067] 7)逆転写 (RT)-PCR [0067] 7) Reverse transcription (RT) -PCR
RT-PCRを行い、 MDA-231細胞における少量のヒト PCIの発現を評価した。オリゴ d Tプライマーおよび Superscript第一鎖 cDNA合成キット（Invitrogen Corp.,カリフオル ユア州、カールズバッド)を用いて、製造業者の説明に従い、 MDA231細胞力も抽出 した全 RNA(5 μ g)を逆転写した。ヒト PCIの増幅に用いたフォワードおよびリバース PC Rプライマーの塩基配列は次のとおりである。配列番号： 7と配列番号： 8で示したブラ イマ一は、それぞれ PCIの cDNA (配列番号： 1)の 733〜752位、および 1289〜127 0位に対応している (Suzuki K., J. Biol. Chem. 262:611-6, 1987)  RT-PCR was performed to evaluate the expression of a small amount of human PCI in MDA-231 cells. Total RNA (5 μg) from which MDA231 cell force was also extracted was reverse transcribed using oligo dT primer and Superscript first strand cDNA synthesis kit (Invitrogen Corp., Carlsbad, Calif.) According to the manufacturer's instructions. The nucleotide sequences of the forward and reverse PCR primers used for human PCI amplification are as follows. The primers shown in SEQ ID NO: 7 and SEQ ID NO: 8 correspond to positions 733 to 752 and positions 1289 to 1270 of PCI cDNA (SEQ ID NO: 1), respectively (Suzuki K., J Biol. Chem. 262: 611-6, 1987)
5'- GAGCAAGACTTCTACGTGAC- 3' (配列番号： 7)および  5'-GAGCAAGACTTCTACGTGAC-3 '(SEQ ID NO: 7) and
5 '-CGGTTCACTTTGCCAAGGAA-3 ' (配列番号： 8) [0068] PCR混合液は、 PCR緩衝液（100 mM Tris- HC1、 200 mM KC1、 pH 8.3)、 25 mM Mg CI、 200 μ Mデォキシリボヌクレオシド（dATP、 dGTP、 dCTP、 dTTP)、それぞれ 0.2 μ5 '-CGGTTCACTTTGCCAAGGAA-3' (SEQ ID NO: 8) [0068] The PCR mixture was composed of PCR buffer (100 mM Tris-HC1, 200 mM KC1, pH 8.3), 25 mM Mg CI, 200 μM deoxyribonucleoside (dATP, dGTP, dCTP, dTTP), respectively. 0.2 μ
2 2
Mのフォワードプライマーおよびリバースプライマー、適量のジェチノレピロカノレボネー ト処理蒸留水、ならびに Taqポリメラーゼ（Roche Biochemicals,スイス、バーゼノレ） 2.5 ユニットから構成された。次いで、 cDNA試料 2 1を含む PCRチューブに混合液を一 定量分注し、 PC-800プログラムテンプコントロールシステム（アステック、 日本、福岡） を用いて増幅を行った。続いて、 PCR産物を 1%ァガロースゲルに電気泳動し、 0.5 g /mlェチジゥムブロマイドで染色した。  It consisted of 2.5 units of M forward primer and reverse primer, an appropriate amount of distilled water treated with Jetinorepyrocanorebonate, and Taq polymerase (Roche Biochemicals, Basenole, Switzerland). Next, a certain amount of the mixture was dispensed into a PCR tube containing cDNA sample 21 and amplified using a PC-800 program temp control system (Astech, Fukuoka, Japan). Subsequently, the PCR product was electrophoresed on a 1% agarose gel and stained with 0.5 g / ml ethidium bromide.
[0069] 8)バキュロウィルス発現系を用いた、原型ヒト PCI、 R354APCI、 degPCIのプラスミド構 築および発現 [0069] 8) Plasmid construction and expression of prototype human PCI, R354APCI, degPCI using baculovirus expression system
以下のプライマー対を用いて、ポリメラーゼ連鎖反応 (PCR)により、シグナルぺプチ ド配列を含まない野生型 PCI cDNAを調製した (Saiki R.K. et al., Science 239:487-91 , 1988)。配列番号： 9および配列番号： 1で示したプライマーは、それぞれ PCIの cDN A (配列番号： 1)の 133〜152位、および 1296〜 1276位に対応している。下線を引 いたヌクレオチドは、挿入された BamH I部位を示す (Hayashi T. et al., Int. J. Hemato 1. 58:213-224, 1993)。  Wild-type PCI cDNA containing no signal peptide sequence was prepared by polymerase chain reaction (PCR) using the following primer pair (Saiki R.K. et al., Science 239: 487-91, 1988). The primers shown in SEQ ID NO: 9 and SEQ ID NO: 1 correspond to positions 133-152 and 1296-1276 of PCI cDNA (SEQ ID NO: 1), respectively. Underlined nucleotides indicate the inserted BamHI site (Hayashi T. et al., Int. J. Hemato 1. 58: 213-224, 1993).
5し GCGGATCCCCACCGCCACCACCCCCGGGA- 3' (配列番号： 9)、および 5し GGCGGATCCTCAGGGGCGGTTCACTTTGCC- 3 ' (配列番号： 10) [0070] シグナルペプチド配列を含まな!/、degPCI cDNAもまた、以下のプライマー対を用い て、 PCRにより調製した。配列番号： 11および配列番号： 12で示したプライマーは、 それぞれ PCIの cDNA (配列番号： 1)の 133〜152位、および 1194〜 1175位に対 応している。下線を引いたヌクレオチドは、挿入された BamH I部位を示す (Hayashi T. et al" Int. J. Hematol. 58:213-224, 1993)。  GCGGATCCCCACCGCCACCACCCCCGGGA-3 '(SEQ ID NO: 9), and 5 GGCGGATCCTCAGGGGCGGTTCACTTTGCC-3' (SEQ ID NO: 10) [0070] No signal peptide sequence! /, DegPCI cDNA also uses the following primer pair: Prepared by PCR. The primers shown in SEQ ID NO: 11 and SEQ ID NO: 12 correspond to positions 133 to 152 and 1194 to 1175 of the PCI cDNA (SEQ ID NO: 1), respectively. Underlined nucleotides indicate the inserted BamHI site (Hayashi T. et al "Int. J. Hematol. 58: 213-224, 1993).
5し GGCGGATCCCCACCGCCACCACCCCCGGGA- 3' (配列番号： 11)、および 5し GGCGGATCCTCACCTGAAAGTGAAGATTGTCC- 3' (配列番号： 12) 増幅した断片を pBluescript SKII(+)の BamH I部位にサブクローユングした後、これら のプラスミドの DNA配列を ABI 310 DNAシーケンサー（ABI、カリフォルニア州、フォス ターシティ一）により確認した。 [0071] 続いて、ミツバチメリチンシグナルペプチドおよび免疫グロブリンの Fc領域との融合 タンパク質を発現させるために、バキュロウィルストランスファーベクター pFastBaclを 用いて構成されたバキュロウィルス発現ベクター pFastBacl- Msp- Fcに (Fujita M. et a 1., Thromb. Res. 105:95-102, 2002)、シグナルペプチドを含まない野生型ヒト PCI cD NAおよび degPCI cDNAを挿入した。シグナルペプチド配列を含まない R354APCI cD NAを含む pBluescript SKII(+)は、次の合成オリゴヌクレオチドプライマーを用い、シグ ナルペプチドを有する R354APCI cDNAを铸型として調製した。配列番号： 13および 配列番号： 14で示したプライマーは、それぞれ PCIの cDNA (配列番号： 1)の 133〜1 52位、および 1296〜 1276位に対応している。下線を引いたヌクレオチドは、挿入さ れた BamH I部位を示す (Hayashi T. et al, Int. J. Hematol. 58:213-224, 1993)。 5) GGCGGATCCCCACCGCCACCACCCCCGGGA-3 '(SEQ ID NO: 11), 5 GGCGGATCCTCACCTGAAAGTGAAGATTGTCC-3' (SEQ ID NO: 12) The DNA sequence was confirmed with an ABI 310 DNA sequencer (ABI, Foster City, CA). [0071] Subsequently, a baculovirus expression vector pFastBacl-Msp-Fc constructed using the baculovirus transfer vector pFastBacl was used to express a fusion protein of the bee melittin signal peptide and the Fc region of immunoglobulin (Fujita M et a 1., Thromb. Res. 105: 95-102, 2002), wild type human PCI cDNA and degPCI cDNA without signal peptide were inserted. PBluescript SKII (+) containing R354APCI cDNA not containing a signal peptide sequence was prepared from the R354APCI cDNA having a signal peptide as a saddle type using the following synthetic oligonucleotide primers. The primers represented by SEQ ID NO: 13 and SEQ ID NO: 14 correspond to positions 133 to 152 and 1296 to 1276 of the PCI cDNA (SEQ ID NO: 1), respectively. Underlined nucleotides indicate the inserted BamHI site (Hayashi T. et al, Int. J. Hematol. 58: 213-224, 1993).
5し GGCGGATCCCCACCGCCACCACCCCCCGGGA- 3' (配列番号： 13)、およ び  5 GGCGGATCCCCACCGCCACCACCCCCCGGGA-3 '(SEQ ID NO: 13), and
5し GGCGGATCCTCAGGGGCGGTTCACTTTGCC- 3 ' (配列番号： 14) DNAの塩基配列を確認した後、シグナルペプチドを含まない適切な R354APCI cD NAを pFastBacl- Msp- Fcの BmH I部位に挿入した。 10% FBSを含むグレース昆虫細 胞培地（Invitrogen Corp.,カリフォルニア州、カールズバッド）で培養した Sf- 9細胞を 用いて、製造業者の説明に従い（Invitrogen Corp.)、原型 PCI、 R354APCI、および de gPCIを発現させるための組換えバキュロウィルスの作製および増幅を行った。無血清 培地 EX- CELL 400 (JRH, BIOSCIENCES,米国、カンザス州、レネクサ）で High- Five 細胞を培養し、これにバキュロウィルスを感染させ、 27°Cで 72時間培養した。  5) GGCGGATCCTCAGGGGCGGTTCACTTTGCC-3 '(SEQ ID NO: 14) After confirming the base sequence of DNA, an appropriate R354APCI cDNA containing no signal peptide was inserted into the BmHI site of pFastBacl-Msp-Fc. Using Sf-9 cells cultured in Grace Insect Cell Medium with 10% FBS (Invitrogen Corp., Carlsbad, Calif.) According to the manufacturer's instructions (Invitrogen Corp.), prototype PCI, R354APCI, and degPCI Recombinant baculovirus for expression was produced and amplified. High-Five cells were cultured in serum-free medium EX-CELL 400 (JRH, BIOSCIENCES, Lenexa, Kansas, USA), infected with baculovirus, and cultured at 27 ° C for 72 hours.
[0072] 9)組換え原型 PCI、 R354APCI、および degPCIの精製 [0072] 9) Purification of recombinant prototype PCI, R354APCI, and degPCI
各組換え PCI (原型 PCI、 R354APCUおよび degPCI)の培養上清を回収し、 10 mM ベンズアミジンの存在下で最終濃度 70%の (NH ) SOを用いて塩析することにより濃縮  Collect the culture supernatant of each recombinant PCI (prototype PCI, R354APCU and degPCI) and concentrate by salting out using 70% (NH) SO in the final concentration in the presence of 10 mM benzamidine.
4 2 4  4 2 4
した。遠心分離後、沈殿物を回収し、 10 mMベンズアミドを含む 50 mM Tris- HC1、 pH 7.5で溶解し、 1 mMベンズアミドを含む 50 mM Tris- HC1、 pH 7.5に対して透析した。 次に、各透析試料を 0.22 μ mフィルター（MILLEX-HA)に通し、ベンズアミドを含まな い同じ緩衝液で平衡化した Hitrap CM FFの 5 mlカラムに供した。平衡緩衝液でカラ ムを洗浄した後、 0 M〜0.5 M NaClの 720 μ 1/分直線勾配でタンパク質を溶出し、 1 m 1画分を回収した。続いて、 PCIを含む画分を収集し、 50 mM NaClを含む Tris_HCl緩 衝液、 pH 7.4に対して透析し、次に同じ緩衝液で平衡化した HiTrapへパリン FFの 1 mlカラムに供した。 0.05 M〜0.5 M NaClの 500 1/分直線勾配でタンパク質を溶出し 、 500 1画分を回収した。以下に記載するように、ウェスタンプロットにより各組換え P CIを検出した。組換え PCIを含む画分を収集し、 VIVASPIN 6 ml CONCENTRATOR ( VIVASCIENCE、ドイツ、ハノーバー）により濃縮および脱塩した。リン酸緩衝食塩水（ PBS)に溶解した精製組換え PCIの一定分割量を、還元条件下でドデシル硫酸ナトリ ゥム-ポリアクリルアミドゲル電気泳動 (SDS- PAGE)に供し、銀染色した。 did. After centrifugation, the precipitate was collected, dissolved in 50 mM Tris-HC1, pH 7.5 containing 10 mM benzamide, and dialyzed against 50 mM Tris-HC1, pH 7.5 containing 1 mM benzamide. Each dialysis sample was then passed through a 0.22 μm filter (MILLEX-HA) and applied to a 5 ml column of Hitrap CM FF equilibrated with the same buffer without benzamide. After washing the column with equilibration buffer, the protein is eluted with a linear gradient of 720 μ1 / min from 0 M to 0.5 M NaCl, 1 m One fraction was collected. Subsequently, fractions containing PCI were collected, dialyzed against Tris_HCl buffer, pH 7.4 containing 50 mM NaCl, and then applied to a 1 ml column of Parin FF to HiTrap equilibrated with the same buffer. The protein was eluted with a linear gradient of 0.05 M to 0.5 M NaCl at 500 1 / min, and 500 1 fractions were collected. Each recombinant PCI was detected by Western plot as described below. Fractions containing recombinant PCI were collected, concentrated and desalted with VIVASPIN 6 ml CONCENTRATOR (VIVASCIENCE, Hannover, Germany). A fixed aliquot of purified recombinant PCI dissolved in phosphate buffered saline (PBS) was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and stained with silver.
[0073] 10) SDSポリアクリルアミド電気泳動 (SDS- PAGE)およびウェスタンブロット解析 [0073] 10) SDS polyacrylamide electrophoresis (SDS-PAGE) and Western blot analysis
SDS- PAGEは Laemmli法 (Laemmli U.K. et al.， Nature 227:680-5, 1970)に従って行 つた。電気泳動した後、ゲル内のタンパク質を-トロセルロース膜に電気的に転写し た。次いで、膜上の原型 PCI、 R354APCUおよび degPCIを、以下のようにウェスタンブ ロット解析により検出した。膜をまず抗 PCIゥサギ IgG (非特許文献 4)で処理し、次に抗 ゥサギ IgGアルカリフォスファターゼコンジュゲートで処理した。ニトロセルロース膜上 のバンドを、以前に記載したようにウェスタンブルー安定ィ匕基質を用いて可視化した  SDS-PAGE was performed according to the Laemmli method (Laemmli U.K. et al., Nature 227: 680-5, 1970). After electrophoresis, the proteins in the gel were electrically transferred to a -trocellulose membrane. Subsequently, prototype PCI, R354APCU and degPCI on the membrane were detected by Western blot analysis as follows. The membrane was first treated with anti-PCI rabbit IgG (Non-Patent Document 4) and then with an anti-rabbit IgG alkaline phosphatase conjugate. Bands on the nitrocellulose membrane were visualized using a Western Blue stable substrate as previously described.
[0074] 11)組換え PCIの活性を測定するアツセィ法 [0074] 11) Atsey method for measuring recombinant PCI activity
様々な組換え PCIの活性は、以下の通りに決定した。 APC(40 g/ml)10 1を、 30 U /mlへパリンを含む TBS 10 μ 1および組換え原型 PCI、 R354APCI,および degPCI(40 μ g/ml O/z 1と混合した。混合物を 37°Cで 20分間インキュベートした後、 0.2 mM S- 2 366、 50 mM Tris- HCl(pH 8.0)、および 0.1 M CsClを含む緩衝液 250 μ 1と混合した。 混合物を室温で 15分間インキュベートした後、 100%酢酸 25 1を添加して反応を停止 した。モデル 550マイクロプレートリーダー（Bio- RAD)を用いて、 405 nmにおける吸光 度を測定した。 APCの段階希釈物を用いて検量線を作成し、残存 APC活性を算出し た。  The activity of various recombinant PCIs was determined as follows. APC (40 g / ml) 101 was mixed with TBS 10 μl containing 30 U / ml heparin and recombinant prototype PCI, R354APCI, and degPCI (40 μg / ml O / z 1. The mixture was 37 After 20 minutes of incubation at ° C, it was mixed with 250 μl of buffer containing 0.2 mM S-2366, 50 mM Tris-HCl (pH 8.0), and 0.1 M CsCl After the mixture was incubated at room temperature for 15 minutes The reaction was stopped by adding 100% acetic acid 251. Absorbance was measured at 405 nm using a model 550 microplate reader (Bio-RAD) A calibration curve was prepared using serial dilutions of APC. Residual APC activity was calculated.
[0075] 12) in vitro浸潤アツセィ法  [0075] 12) In vitro infiltration method
Albini(Albini A. et al.， Cancer Res. 47:3239-45, 1987)によって記載されている通り に、 MDA-231細胞の侵襲性に関するアツセィを行った。簡潔に説明すると、 Engelbre th- Holm- Swarm(EHS)マウス肉腫から抽出されたマトリゲル基底膜マトリックスを 25 μ g /フィルターでコーティングしたトラックエッチングポリエチレンテレフタラート (PET)メン ブレンフィルター（8 μ mポアサイズ）を備えた直径 6.4 mmのトランスゥエル（Becton Die kinson、カリフォルニア州、マウンテンビュー）を使用した。ー晚乾燥させた後、 8 m ポリカーボネート膜を含む細胞培養インサートを、無血清 DMEM中、 37°Cで 2時間膨 潤させた。野生型、原型 PCI、 R354APCI、 degPCI、または Mock発現 MDA-231細胞株 をトリプシン処理により回収し、 FBSおよびプロティナーゼ阻害剤を含まない DMEMで 洗浄した。 An assessment of the invasiveness of MDA-231 cells was performed as described by Albini (Albini A. et al., Cancer Res. 47: 3239-45, 1987). In brief, Engelbre 6.4 mm diameter with a track-etched polyethylene terephthalate (PET) membrane filter (8 μm pore size) coated with 25 μg / filter Matrigel basement membrane matrix extracted from th-Holm- Swarm (EHS) mouse sarcoma Transwell (Becton Die kinson, Mountain View, CA) was used. -After drying, the cell culture insert containing 8 m polycarbonate membrane was swollen in serum-free DMEM at 37 ° C for 2 hours. Wild-type, prototype PCI, R354APCI, degPCI, or Mock-expressing MDA-231 cell lines were harvested by trypsin treatment and washed with DMEM without FBS and proteinase inhibitors.
[0076] 次いで、精製した PCI、 R354APCI、 degPCI、 uPA (Technocloneゝオーストリア、ウイ一 ン）の存在下または非存在下において、 FBSを含まない DMEM 500 1中に細胞（2 x 105)を懸濁し、培養インサート (上チャンバ一）に入れた。下チャンバ一を、化学誘引 物質として用いる 10% FBSを含む DMEM 750 1で満たした。 5% CO下で 37°Cで 24時 [0076] The cells (2 x 10 5 ) were then suspended in DMEM 500 1 without FBS in the presence or absence of purified PCI, R354APCI, degPCI, uPA (Technoclone ゝ Austria, Winn). It became turbid and placed in the culture insert (upper chamber 1). The lower chamber was filled with DMEM 750 1 containing 10% FBS used as a chemoattractant. 24 hours at 37 ° C under 5% CO
2  2
間インキュベートした後、各インサートの膜の上表面を綿棒で穏やかにこすり、非浸 潤細胞およびマトリゲルをすベて除去した。膜の下表面上の浸潤細胞を、 Diff-Quik 染色キット（国際試薬、日本、神戸)を用いて固定し染色した。光学顕微鏡下で 100 X 倍率において、細胞をカウントした。  After an inter-incubation, the upper surface of each insert membrane was gently rubbed with a cotton swab to remove any non-soaked cells and Matrigel. Infiltrating cells on the lower surface of the membrane were fixed and stained using the Diff-Quik staining kit (International Reagent, Kobe, Japan). Cells were counted at 100X magnification under a light microscope.
[0077] 13)動物 [0077] 13) Animals
in vivoにおける様々な MDA-231細胞の転移能および増殖を評価するため、雄およ び雌の重症複合免疫不全マウス (SCID) (5週齢)を日本クレア（日本、大阪)から購入 した。マウスは、 12時間明 /12時間暗の一定サイクルで飼育し、標準的な食餌および 水を自由に得られるようにした。実験は三重大学動物実験審査委員会により承認さ れ、国立保健研究所の実験動物指針に従って行った。  To assess the metastatic potential and proliferation of various MDA-231 cells in vivo, male and female severe combined immunodeficient mice (SCID) (5 weeks old) were purchased from CLEA Japan (Osaka, Japan). Mice were housed in a constant cycle of 12 hours light / 12 hours dark, allowing free access to standard food and water. The experiment was approved by the Mie University Animal Experiments Review Committee and conducted in accordance with the laboratory animal guidelines of the National Institutes of Health.
[0078] 14) in vivoにおける腫瘍増殖の評価 [0078] 14) Evaluation of tumor growth in vivo
滅菌した DMEM 0.2 ml中の MDA— PCI、 MDA— R354APCI、 MDA— degPCI、または M DA-Mock細胞（5 x 105細胞）を、 5週齢の雄 SCIDマウスの側腹部に皮内注射した。 週に 1度ノギスを用いて腫瘍の大きさを測定し、腫瘍増殖をモニターした。以下の式 に従って腫瘍容積を決定した: V = (L X W2) x 0.52、式中、 Lは長さであり Wは幅であ る。 [0079] 15)実験的肺転移のアツセィ法 MDA-PCI, MDA-R354APCI, MDA-degPCI, or MDA-Mock cells (5 × 10 5 cells) in 0.2 ml of sterile DMEM were injected intradermally into the flank of 5 week old male SCID mice. Tumor size was measured once a week using calipers and tumor growth was monitored. Tumor volume was determined according to the following formula: V = (LXW 2 ) x 0.52, where L is the length and W is the width. [0079] 15) Atsy method of experimental lung metastasis
サブコンフルェントな MDA- PCI、 MDA-R354APCU MDA- degPCI、または MDA- Mo ck細胞を EDTA溶液を用いて回収し、無血清 DMEMを用いて適切な密度（2.5 x 106 細胞/ ml)に再懸濁した。次いで、 MDA-PCU MDA-R354APCI, MDA- degPCI、また は MDA-Mock細胞を、雌 SCIDマウスの尾静脈に注射した。腫瘍を注射して力も 35日 後、ペントバルビタールを用いてマウスを麻酔し、屠殺した。肺を摘出し、ホルムアル デヒド中性緩衝液中で固定した。拡大鏡を用いて、白い斑点として見える小塊をカウ ントした。 Subconfluent E cement of MDA- PCI, MDA-R354APCU MDA- degPCI , or MDA Mo ck cells were harvested with EDTA solution, the appropriate density (2.5 x 10 6 cells / ml) with serum-free DMEM re Suspended. MDA-PCU MDA-R354APCI, MDA-degPCI, or MDA-Mock cells were then injected into the tail vein of female SCID mice. Thirty-five days after tumor injection, the mice were anesthetized with pentobarbital and sacrificed. Lungs were removed and fixed in formaldehyde neutral buffer. Using a magnifying glass, a small blob that appeared as white spots was counted.
[0080] 16)マトリゲル移植アツセィ法  [0080] 16) Matrigel transplantation Atsey method
以前に記載されているように (McMahon G.A. et al.， J. Biol. Chem. 276:33964-68, 2001)、 MDA- PCI、 MDA- R354APCI、 MDA- degPCI、または MDA- Mock細胞（2 x 106 細胞/ ml)、 VEGF、および様々な濃度のへパリンを含むマトリゲル 0.5 ml (9〜10 mg/ ml ; Becton Diskinson、ニュージャージー州、フランクリンレイク）を、各雄 SCIDマウス の腹部正中に皮下注射した。 3日後、マトリゲルプラグに向力 毛細血管を、デジタル カメラシステム（Olympus,ニューヨーク州、メルビル）を用いて可視化した。平行した 実験では、ハンクス液に溶解した 0.1%コラゲナーゼ 1 mlでマトリゲルプラグを消化し、 ヘモグロビンアツセィ法 (Passaniti A. et al.， Lab. Invest. 67:519-28, 1992)により新た な血管の数を定量した。 As previously described (McMahon GA et al., J. Biol. Chem. 276: 33964-68, 2001), MDA-PCI, MDA-R354APCI, MDA-degPCI, or MDA-Mock cells (2 x 10 6 cells / ml), VEGF, and 0.5 ml of Matrigel (9-10 mg / ml; Becton Diskinson, Franklin Lake, NJ) with various concentrations of heparin, subcutaneously in the midline of the abdomen of each male SCID mouse did. Three days later, centripetal capillaries on Matrigel plugs were visualized using a digital camera system (Olympus, Melville, NY). In parallel experiments, the Matrigel plug was digested with 1 ml of 0.1% collagenase dissolved in Hank's solution, and a new blood vessel was obtained using the hemoglobin atsey method (Passaniti A. et al., Lab. Invest. 67: 519-28, 1992). The number of was quantified.
[0081] 17)二ヮトリ漿尿膜 (CAM)アツセィ法  [0081] 17) Two-tailed chorioallantoic membrane (CAM) Atsey method
環を備えた開口プラスチックチューブ（直径 8 cm,後部 6 cm)に正方形のプラスチッ クラップを固定し、卵用ハンモックを準備した。受精卵（3日齢)を割り、内容物を卵用 ハンモックに移し、胚が卵の表面に来るように操作した。無菌性を維持するために、 ハンモックの上にペトリ皿の蓋を被せた。 37°Cの加湿インキュベーター内で、胚を維 持した。フード内で、 2%メチルセルロース 50 μ 1をペトリ皿上でー晚乾燥させ、メチルセ ルロースディスクを調製した。皿カゝらディスクを剥がし、 CAM上に載せ、次いで原型 P CI、 R354APCUまたは degPCIをディスク上に添カ卩した。デジタルカメラシステム（Olym pus、ニューヨーク州、メルビル）および NIH Image 1.61 (NIH、メリーランド州、ベテスダ )により、 CAMの画像を撮影した。倍率 X 100のランダムな 6視野にある無傷の血管の 数をカウントした。 A square plastic wrap was fixed to an open plastic tube with a ring (diameter 8 cm, rear 6 cm) to prepare an egg hammock. The fertilized egg (3 days old) was divided, the contents were transferred to an egg hammock, and the embryo was manipulated so that it was on the surface of the egg. In order to maintain sterility, a Petri dish lid was placed over the hammock. Embryos were maintained in a 37 ° C humidified incubator. In a hood, 2% methylcellulose 50 μl was dried on a Petri dish to prepare a methylcellulose disk. The disc was peeled off from the pan and placed on the CAM, and then the prototype PCI, R354APCU or degPCI was added onto the disc. CAM images were taken with a digital camera system (Olympus, Melville, NY) and NIH Image 1.61 (NIH, Bethesda, MD). Of 100x random, unaffected blood vessels in 6 fields of view Counted the number.
[0082] 18) in vitro血管新生アツセィ法  [0082] 18) In vitro angiogenesis method
以前に記載されている通りに（Schnaper H.W. et al.， J. Cell Physiol. 165:107-18, 1 995)、マトリゲル上での毛管形成アツセィを行った。 24ゥエルプレートにマトリゲル（Be cton Dickinson,ニュージャージー州、フランクリンレイク）を 4°Cでコーティングし、 37 °Cで 30分間重合させた。マトリゲルコーティングプレートに、 HUVECを播種した（2 X 1 04細胞/ゥエル）。へパリンの非存在下において、細胞を原型 PCI、 R354APCIもしくは degPCI (10 μ g/ml)と共に、またはこれらの PCIの非存在下で (対照）、インキュベート した。インキュベーションしてから 6時間後、位相差顕微鏡下で毛管形成を視覚的に 評価した。倍率 X 100のランダムな 6視野にある無傷の管の数をカウントした。 Capillary assembly on matrigel was performed as previously described (Schnaper HW et al., J. Cell Physiol. 165: 107-18, 1 995). A 24 well plate was coated with Matrigel (Becton Dickinson, Franklin Lake, NJ) at 4 ° C and polymerized at 37 ° C for 30 minutes. Matrigel-coated plates were seeded with HUVEC (2 × 10 4 cells / well). In the absence of heparin, cells were incubated with prototypical PCI, R354APCI or degPCI (10 μg / ml) or in the absence of these PCIs (control). After 6 hours of incubation, capillary formation was assessed visually under a phase contrast microscope. The number of intact tubes in 6 random fields with a magnification of X100 was counted.
[0083] 19)統計解析  [0083] 19) Statistical analysis
値はすべて、平均値士平均値の標準偏差として表した。どの実験も、少なくとも 3回 繰り返した。分散分析を用いて、有意な相違を評価した。 Pく 0.05の値を、統計学的に 有意であると見なした。  All values were expressed as the standard deviation of the average value. Each experiment was repeated at least 3 times. Analysis of variance was used to assess significant differences. A value of 0.05 was considered statistically significant.
[0084] く結果〉  [0084] <Result>
〔実施例 1〕バキュロウィルス発現系により産生された組換え原型 PCI、 R354APCI、お よび degPCIの発現、精製、および APC阻害活性  [Example 1] Expression, purification, and APC inhibitory activity of recombinant prototype PCI, R354APCI, and degPCI produced by baculovirus expression system
バキュロウィルス発現系により発現された原型 PCI、 R354APCI、および degPCIを、 Hi trap CM FFおよび Hitrapへパリン FFを用いて精製した。次いで、精製したタンパク質 を、還元条件下でのウェスタンブロット解析により検出した。原型 PCI、 R354APCI、お よび degPCIの MWは、それぞれ約 57 KDa,約 57 KDa,および約 54 KDaであった（図 1 )。これらの組換え PCIの APC阻害活性についても試験した。 R354APCIおよび degPCI は、 APCを阻害することがほとんどできな力つた（図 2)。  Prototype PCI, R354APCI and degPCI expressed by baculovirus expression system were purified using Hi trap CM FF and Hitrap heparin FF. The purified protein was then detected by Western blot analysis under reducing conditions. The MW of the prototype PCI, R354APCI, and degPCI were approximately 57 KDa, approximately 57 KDa, and approximately 54 KDa, respectively (Figure 1). These recombinant PCIs were also tested for APC inhibitory activity. R354APCI and degPCI were almost unable to inhibit APC (Figure 2).
[0085] 〔実施例 2〕 MDA-231細胞の侵襲性に及ぼす PCI、抗ヒト uPA抗体、 PAI-1、および uP Aの効果 [Example 2] Effects of PCI, anti-human uPA antibody, PAI-1, and uPA on the invasiveness of MDA-231 cells
マトリゲルシステムにおける、 MDA-231細胞の侵襲性に及ぼす原型 PCIの効果。ま ず、 ELISAおよび RT-PCR解析により、 MDA-231細胞での uPAおよび PCI発現を評価 した。 MDA-231細胞は、それぞれ 470 ng/104細胞 /24時間の uPA、および 58 ng/104 細胞 /24時間の PCIを発現する。 RT-PCR解析力 も、 MDA-231細胞により uPA mRN Aおよび PCI mRNAの両方が発現することが示された（データは示さず)。続いて、 MD A-231細胞の浸潤活性に及ぼす PCIの効果を試験した。原型 PCIは MDA-231細胞の 浸潤活性を用量依存的にかつ有意に阻害したが（図 3A)、 BSAにはこの活性に対す る効果がな力つた (データは示さず)。さらに、抗ヒト PCI抗体は、 MDA-231細胞浸潤 の PCI誘導性阻害を用量依存的に阻止した (データは示さず)。浸潤アツセィにより、 MDA-231細胞の侵襲性が抗 uPA抗体（図 3B)および PAI-1 (図 3C)によって阻害され 、 uPAによって用量依存的に促進される（図 3D)ことも示された。 Effect of prototype PCI on the invasiveness of MDA-231 cells in the Matrigel system. First, uPA and PCI expression in MDA-231 cells was evaluated by ELISA and RT-PCR analysis. MDA-231 cells are 470 ng / 10 4 cells / 24 h uPA, respectively, and 58 ng / 10 4 Cells / 24 hours of PCI expression. RT-PCR analysis also indicated that both uPA mRNA and PCI mRNA were expressed by MDA-231 cells (data not shown). Subsequently, the effect of PCI on the invasive activity of MDA-231 cells was tested. Prototype PCI significantly inhibited the invasion activity of MDA-231 cells in a dose-dependent manner (Figure 3A), but BSA was potent in effect on this activity (data not shown). Furthermore, anti-human PCI antibodies blocked PCI-induced inhibition of MDA-231 cell invasion in a dose-dependent manner (data not shown). Invasion assay also showed that the invasiveness of MDA-231 cells was inhibited by anti-uPA antibodies (Figure 3B) and PAI-1 (Figure 3C) and promoted by uPA in a dose-dependent manner (Figure 3D).
[0086] 〔実施例 3〕 MDA-231細胞の侵襲性に及ぼす原型 PCI、 R354APCI、および degPCIの 効果 [0086] [Example 3] Effect of prototype PCI, R354APCI, and degPCI on invasiveness of MDA-231 cells
MDA-231細胞の侵襲性に対する PCIの阻害活性力PCIのプロテアーゼ阻害活性に 依存するのかどうかを評価するため、 MDA-231細胞の侵襲性に及ぼす原型 PCI、 R3 54APCI、および degPCIの効果を評価した。図 4に示すように、原型 PCIは MDA-231細 胞の侵襲性を有意に阻害した力 R354APCIも degPCIも MDA-231細胞の浸潤を阻害 しなかった。これらの知見から、 MDA-231細胞の in vitro侵襲性を阻害するためには 、 PCIのプロテアーゼ阻害活性が必要であることが示唆される。  In order to evaluate whether PCI's inhibitory activity on MDA-231 cell invasiveness depends on the protease inhibitory activity of PCI, the effects of prototype PCI, R3 54APCI, and degPCI on the invasiveness of MDA-231 cells were evaluated . As shown in Figure 4, the prototype PCI significantly inhibited the invasiveness of MDA-231 cells. Neither R354APCI nor degPCI inhibited the invasion of MDA-231 cells. These findings suggest that PCI's protease inhibitory activity is required to inhibit the in vitro invasiveness of MDA-231 cells.
[0087] 〔実施例 4〕それぞれの発現ベクターをトランスフエクシヨンした MDA-231細胞におけ る原型 PCI、 R354APCUまたは degPCIの発現 [Example 4] Expression of prototype PCI, R354APCU, or degPCI in MDA-231 cells transfected with the respective expression vectors
原型 PCI、 R354APCI、および degPCIの発現が MDA-231細胞の侵襲性、増殖、およ び転移能に影響を及ぼすカゝどうかを評価するため、 2つの PCI発現 MDA-231細胞株 ( MDA-PCI 1、 MDA-PCI 2)、 2つの変異体 PCI発現 MDA- 231細胞株（MDA- R354AP CI 1、 MDA-R354APCI 2)、 2つの degPCI発現 MDA- 231細胞株（MDA- degPCI 1、 M DA- degPCI 2)、および 2つの Mockトランスフエクシヨン細胞株（MDA- Mock 1、 MDA- Mock 2)を調製した。 MDA- PCI、 MDA- R354APCI、および MDA- degPCI細胞株は各 PCI mRNAの強力な発現を示したのに対し、トランスフエクシヨンしていない MDA細胞 および MDA- Mock細胞株は原型 PCIを弱く発現して!/、た（データは示さず)。 MDA-P CI 1および MDA-PCI 2細胞により分泌される PCIの量は、それぞれ 10.3および 12.5 n g/104細胞 /24時間であった。 MDA- R354APCI 1および MDA- R354APCI 2細胞によ り分泌される R354APCIの量は、それぞれ 20.0および 12.0 ng/104細胞 /24時間であつ た。 MDA- degPCI 1および MDA- degPCI 2細胞より分泌される分解型 PCIの量は、そ れぞれ 3.0および 1.1 ng/104細胞 /24時間であった。これらの細胞株の増殖速度およ び uPA産生は、ほぼ同じであった（データは示さず)。 To evaluate whether the expression of prototype PCI, R354APCI, and degPCI affects the invasiveness, proliferation, and metastatic potential of MDA-231 cells, two PCI-expressing MDA-231 cell lines (MDA-PCI 1, MDA-PCI 2), two mutant PCI expression MDA-231 cell lines (MDA-R354AP CI 1, MDA-R354APCI 2), two degPCI expression MDA-231 cell lines (MDA-degPCI 1, MDA- degPCI 2) and two Mock transfection cells lines (MDA-Mock 1 and MDA-Mock 2) were prepared. MDA-PCI, MDA-R354APCI, and MDA-degPCI cell lines showed strong expression of each PCI mRNA, whereas untransfected MDA and MDA-Mock cell lines expressed weakly the prototype PCI. / !, (data not shown). The amount of PCI secreted by MDA-P CI 1 and MDA-PCI 2 cells was 10.3 and 12.5 ng / 10 4 cells / 24 hours, respectively. MDA-R354APCI 1 and MDA-R354APCI 2 cells The amount of R354APCI secreted was 20.0 and 12.0 ng / 10 4 cells / 24 hours, respectively. The amounts of degraded PCI secreted from MDA-degPCI 1 and MDA-degPCI 2 cells were 3.0 and 1.1 ng / 10 4 cells / 24 hours, respectively. The growth rate and uPA production of these cell lines were almost the same (data not shown).
[0088] 〔実施例 5〕 MDA-231細胞の in vitro侵襲性に及ぼす原型 PCI、 R354APCI,または de gPCI発現の効果 [0088] [Example 5] Effect of prototype PCI, R354APCI, or degPCI expression on in vitro invasiveness of MDA-231 cells
原型 PCI、 R354APCI、または degPCIの発現が MDA-231細胞の浸潤能に影響を及 ぼすかどうかを判定するため、マトリゲルにおける PCI、 R354APCI、 degPCI発現 MDA -231細胞および Mockトランスフエクシヨン MDA-231細胞の侵襲性を in vitroで評価し た。図 5に記載したように、 2つの MDA-PCI細胞株の侵襲性は、 MDA-Mock細胞株よ りも有意に低かった。 MDA-R354APCIおよび MDA-degPCI細胞株の侵襲性は、 MDA -Mock細胞株と有意に異なっていなかった。これらの知見から、 MDA-231細胞で発 現した PCIが浸潤を抑制すること、および MDA-231細胞の浸潤に対する PCIの阻害 活性がそのプロテアーゼ阻害活性に依存して 、ることが示唆される。これらのデータ は、上記の MDA-231細胞の侵襲性に及ぼす組換え R354APCIおよび degPCIの効果 と一致する。  To determine whether the expression of prototype PCI, R354APCI, or degPCI affects the invasion ability of MDA-231 cells, PCI, R354APCI, degPCI expressing MDA-231 cells and Mock transfection MDA-231 cells in Matrigel In vitro invasiveness was evaluated. As described in FIG. 5, the invasiveness of the two MDA-PCI cell lines was significantly lower than that of the MDA-Mock cell line. The invasiveness of the MDA-R354APCI and MDA-degPCI cell lines was not significantly different from the MDA-Mock cell line. These findings suggest that PCI expressed in MDA-231 cells suppresses invasion, and that PCI's inhibitory activity against MDA-231 cell invasion depends on its protease inhibitory activity. These data are consistent with the effects of recombinant R354APCI and degPCI on the invasiveness of MDA-231 cells described above.
[0089] 〔実施例 6〕 MDA-231細胞の増殖に及ぼす原型 PCI、 R354APCI、または degPCIの効 果  [Example 6] Effect of prototype PCI, R354APCI, or degPCI on proliferation of MDA-231 cells
腫瘍増殖に及ぼす PCIの in vivo効果を評価するため、 SCIDマウスの背側に移植し た Mock、 PCI、 R354APCI、または degPCI発現 MDA-231細胞の増殖をモニターした。 図 6に示すように、 MDA-PCI細胞株の増殖は、 Mock発現細胞株よりも有意に低かつ た。驚いたことに、 MDA- R354APCIおよび MDA- degPCI細胞株の増殖もまた、 MDA- Mock細胞株よりも有意に低ぐ MDA-PCIの増殖と同じかまたはそれよりもわずかに低 かった。これらの知見から、 PCIが in vivoで腫瘍増殖を阻害すること、および PCIのこ の増殖阻害活性がそのプロテアーゼ阻害活性に依存しないことが示唆される。  To assess the in vivo effect of PCI on tumor growth, the growth of Mock, PCI, R354APCI, or degPCI expressing MDA-231 cells transplanted on the dorsal side of SCID mice was monitored. As shown in FIG. 6, the growth of the MDA-PCI cell line was significantly lower than that of the Mock expressing cell line. Surprisingly, the growth of the MDA-R354APCI and MDA-degPCI cell lines was also the same or slightly lower than that of the MDA-PCI, which was significantly lower than the MDA-Mock cell line. These findings suggest that PCI inhibits tumor growth in vivo and that this growth inhibitory activity of PCI is independent of its protease inhibitory activity.
[0090] 〔実施例 7〕 MDA-231細胞の転移能に及ぼす原型 PCI、 R354APCI、または degPCIの 効果 [Example 7] Effect of prototype PCI, R354APCI, or degPCI on metastatic potential of MDA-231 cells
MDA-Mock, MDA-PCI, MDA- R354APCI、または MDA- degPCIを尾静脈から注射 した後、転移性肺小塊の数をカウントすることにより、 MDA-231細胞の転移能に及ぼ す PCIの効果を試験した。図 7Aおよび 7Bに示すように、 MDA-PCU MDA-R354APCI 、または MDA-degPCI細胞株の肺小塊の数は、 MDA- Mock細胞株のものよりも有意 に少なかった。さらに、 MDA-R354APCIおよび MDA-degPCI細胞株の転移性小塊は 、 MDA-PCI細胞株のものよりもわずかに大きかった。これらのデータから、 PCIが in vi voで腫瘍転移を阻害すること、および PCIの腫瘍転移に対する阻害活性がそのプロ テアーゼ阻害活性に依存しな 、ことが実証される。 MDA-Mock, MDA-PCI, MDA-R354APCI, or MDA-degPCI injected through the tail vein Thereafter, the effect of PCI on the metastatic ability of MDA-231 cells was examined by counting the number of metastatic lung nodules. As shown in FIGS. 7A and 7B, the number of lung nodules in the MDA-PCU MDA-R354APCI or MDA-degPCI cell lines was significantly less than that in the MDA-Mock cell lines. Furthermore, the metastatic nodules of the MDA-R354APCI and MDA-degPCI cell lines were slightly larger than those of the MDA-PCI cell line. These data demonstrate that PCI inhibits tumor metastasis in vivo and that PCI's inhibitory activity against tumor metastasis is independent of its protease inhibitory activity.
[0091] 〔実施例 8〕 in vivo血管新生に及ぼす原型 PCI、 R354APCI、または degPCIの効果  [0091] [Example 8] Effect of prototype PCI, R354APCI, or degPCI on in vivo angiogenesis
VEGF、へパリン、および原型 PCI、 R354APCU degPCI、または Mock発現 MDA- 231 細胞を含むマトリゲルを皮下注射した動物を用いて、 VEGF誘導性血管新生に及ぼ す PCIの効果を試験した。図 8Aから、 PCI発現 MDA-231細胞ならびに R354APCIおよ び degPCI発現 MDA-231細胞を含むマトリゲルへの血管形成が、 Mock発現 MDA-23 1細胞よりも低かったことが示される。図 8Bに示すように、 MDA-PCI細胞、 MDA-R354 APCIまたは MDA-degPCI細胞株を含むマトリゲル中のヘモグロビンレベルは、 MDA- Mock細胞株のレベルよりも有意に低かった。続いて、 in vivoでの-ヮトリ CAMアツセ ィ法により、様々な組換え PCIの抗血管新生活性を示した。図 9に示すように、 PCIは -ヮトリ CAMアツセィにおいて血管の増殖を有意に阻害した。 PCI処理した CAMは対 照よりも有意に小さぐ血管新生も少な力つた。 R354APCIおよび degPCIもまた、血管 の増殖を有意に阻害した。これらのデータから、 PCIが in vivoで血管新生を阻害する こと、およびこの阻害活性がそのプロテアーゼ阻害活性に依存しないことが示される 。この結果は、 in vivoアツセィで記載した観察と一致する。  The effect of PCI on VEGF-induced angiogenesis was tested using animals injected subcutaneously with VEGF, heparin, and prototype PCI, R354APCU degPCI, or Matrigel containing MDA-231 cells expressing Mock. FIG. 8A shows that angiogenesis on Matrigel containing PCI-expressing MDA-231 cells and R354APCI and degPCI-expressing MDA-231 cells was lower than Mock-expressing MDA-23 1 cells. As shown in FIG. 8B, hemoglobin levels in Matrigel containing MDA-PCI cells, MDA-R354 APCI or MDA-degPCI cell lines were significantly lower than those of MDA-Mock cell lines. Subsequently, various in vivo anti-angiogenic activities of recombinant PCI were demonstrated by in-vivo CAM assay. As shown in FIG. 9, PCI significantly inhibited vascular growth in the ヮ -bird CAM assembly. PCI-treated CAMs were significantly smaller than controls and had less angiogenesis. R354APCI and degPCI also significantly inhibited vascular growth. These data indicate that PCI inhibits angiogenesis in vivo and that this inhibitory activity is independent of its protease inhibitory activity. This result is consistent with the observations described in the in vivo accessory.
[0092] 〔実施例 9〕 HUVECによる管形成に及ぼす原型 PCI、 R354APCI,または degPCIの効 果  [0092] [Example 9] Effect of prototype PCI, R354APCI, or degPCI on tube formation by HUVEC
HUVECの in vitro血管新生に及ぼす様々な PCIの効果を評価した。図 10Aおよび 10 Bに示すように、原型 PCI、 R354APCI、および degPCIにより管形成が阻害された。さら に、抗ヒト PCI抗体は、管形成の PCI誘導性阻害を用量依存的に阻止した (データは 示さず)。興味深いことに、 R354APCIおよび degPCIの血管新生阻害活性は、原型 PC Iよりも有意に強かった。これらのデータから、皮下に移植した R354APCほたは degPC I発現 MDA-231細胞力 原型 PCI発現 MDA-231細胞よりもゆっくりと増殖する理由が 説明され得る。 The effects of various PCIs on in vitro angiogenesis of HUVEC were evaluated. As shown in FIGS. 10A and 10B, tube formation was inhibited by prototype PCI, R354APCI, and degPCI. In addition, anti-human PCI antibodies blocked PCI-induced inhibition of tube formation in a dose-dependent manner (data not shown). Interestingly, the anti-angiogenic activity of R354APCI and degPCI was significantly stronger than the prototype PC I. From these data, R354APC transplanted subcutaneously is degPC The reason why I-expressing MDA-231 cell power proliferates more slowly than prototype PCI-expressing MDA-231 cells may be explained.
[0093] く考察〉 [0093] <Consideration>
最近になって、本発明者らは、非腫瘍性腎組織と比較して腎癌細胞において PCI 発現が有意に減少して 、ること、および PCI発現が腎癌細胞株 Caki-1細胞の浸潤活 性を阻害することを実証した (Wakita T. et al.， Int. J. Cancer 108:516-23, 2004)。ま た、 PCI発現 Caki-1細胞を用いて、腫瘍増殖および転移に及ぼす PCIの効果を in viv oで評価した力 野生型 Caki-1細胞でさえ SCIDマウスで増殖し得な力つた。これに基 づき、乳癌細胞株 MDA-231細胞の侵襲性に及ぼす PCIの効果を試験した。 MDA-23 1細胞は、大量の uPAおよび少量の PCIを発現して!/ヽた。  Recently, the inventors have shown that PCI expression is significantly reduced in renal cancer cells compared to non-neoplastic renal tissue, and that PCI expression is infiltrating the renal cancer cell line Caki-1 cells. It has been demonstrated to inhibit activity (Wakita T. et al., Int. J. Cancer 108: 516-23, 2004). Moreover, PCI-expressing Caki-1 cells were used to assess the effect of PCI on tumor growth and metastasis in vivo. Even wild-type Caki-1 cells were unable to grow in SCID mice. Based on this, the effect of PCI on the invasiveness of breast cancer cell line MDA-231 cells was tested. MDA-23 1 cells expressed large amounts of uPA and small amounts of PCI!
[0094] 精製 PCIは MDA- 231細胞の浸潤活性を効率的かつ有意に阻害した力 PCIの阻害 活性は Caki細胞の浸潤の場合と比較して弱力つた。この結果はおそらぐ Caki-1細 胞よりも非常に高い、 MDA-231による uP発現 Aに起因する。さらに、 MDA-231細胞の 浸潤活性は uPAの添カ卩により増大し、 PAI-1により阻害された。この結果は、 MDA-23 1の浸潤は主として uPAにより媒介されるという考えと一致する。また本発明者らは、 B 16マウスメラノーマ細胞の浸潤活性に及ぼす PCIの効果を評価し、同様の結果を見 出した (データは示さず)。これらの知見から、様々な腫瘍細胞の浸潤が PCIにより制 御されることが示唆される。 [0094] Purified PCI efficiently and significantly inhibited invasive activity of MDA-231 cells. PCI inhibitory activity was weaker than that of Caki cells. This result is probably due to uP expression A by MDA-231, which is much higher than the Caki-1 cells. Furthermore, the invasive activity of MDA-231 cells was increased by uPA supplementation and inhibited by PAI-1. This result is consistent with the notion that MDA-23 1 infiltration is primarily mediated by uPA. The present inventors also evaluated the effect of PCI on the invasive activity of B 16 mouse melanoma cells and found similar results (data not shown). These findings suggest that the invasion of various tumor cells is controlled by PCI.
さらに、本発明者らはいくつかの組換え変異体 PCIを調製して、 MDA-231細胞浸潤 に及ぼすそれらの阻害活性を評価し、原型 PCIが MDA-231細胞の浸潤活性を阻害 することを見出した。 R354APCI (反応部位変異体)およびプロテアーゼ分解型 PCIの N末端断片は、 APCに対する阻害活性を有さない。このことから、 in vitro腫瘍細胞浸 潤に対する PCIの阻害活性がそのプロテアーゼ阻害活性に依存することが示唆され る。  In addition, we prepared several recombinant mutant PCIs and evaluated their inhibitory activity on MDA-231 cell invasion, and demonstrated that prototype PCI inhibits MDA-231 cell invasion activity. I found it. R354APCI (reactive site variant) and the N-terminal fragment of protease-degraded PCI have no inhibitory activity against APC. This suggests that the inhibitory activity of PCI on in vitro tumor cell infiltration depends on its protease inhibitory activity.
[0095] 続いて、本発明者らは、 PCI発現 MDA-231細胞が Mockトランスフエクシヨン MDA-23 1細胞よりも浸潤性が低いことを見出した。この知見は、 PCI発現 Caki-1細胞が Mockト ランスフエクシヨン Cali-1細胞よりも浸潤性が低いことを示す以前のデータと一致する 。本発明者らはまた、 MDA-231細胞の浸潤能に及ぼす PCIのプロテアーゼ阻害活性 の効果を試験し、 degPCI発現 MDA-231細胞および R354APCI発現 MDA-231細胞の 浸潤能が、 Mock発現 MDA-231細胞と有意に違いがないことを実証した。この結果は 、組換ぇ1¾54 ？じ1ぉょび(16§?じ1が ^1"0で\10 -231細胞浸潤に効果を及ぼさな V、ことを示すデータと一致する。 Subsequently, the present inventors have found that PCI-expressing MDA-231 cells are less invasive than Mock transfection MDA-23 1 cells. This finding is in agreement with previous data showing that PCI-expressing Caki-1 cells are less invasive than Mock Transfection Cali-1 cells. The present inventors also described PCI protease inhibitory activity on the invasion ability of MDA-231 cells. The invasive ability of degPCI-expressing MDA-231 cells and R354APCI-expressing MDA-231 cells was not significantly different from that of Mock-expressing MDA-231 cells. This result is a recombination 1¾54? This is consistent with the data showing that 16 § 1 is ^ 1 "0 and has no effect on the invasion of \ 10 -231 cells.
[0096] 次に、様々な PCI発現 MDA-231細胞を用いて、 SCIDマウスにおける MDA-231細胞 の増殖および転移に及ぼす PCIの in vivo効果を試験した。データから、 PCIが腫瘍増 殖および転移の両方を in vivoで阻害することが示された。驚いたことに、 R354APCI および degPCIもまた、腫瘍増殖および転移に対する強力な阻害活性を示し、このこと から、腫瘍増殖および転移に対する PCIの阻害活性がそのプロテアーゼ阻害活性に よって媒介されな 、ことが示唆された。  [0096] Next, various PCI-expressing MDA-231 cells were used to test the in vivo effects of PCI on the proliferation and metastasis of MDA-231 cells in SCID mice. Data showed that PCI inhibits both tumor growth and metastasis in vivo. Surprisingly, R354APCI and degPCI also showed potent inhibitory activity against tumor growth and metastasis, suggesting that the inhibitory activity of PCI against tumor growth and metastasis is not mediated by its protease inhibitory activity. It was done.
[0097] カリスタチン (kallistatin)は、血管新生の抑制を介して腫瘍増殖を阻害するへパリン 結合セルピンである (Miao R.Q. et al.， Blood 100:3245-52, 2002)。 VEGFおよび bFG Fが血管新生の強力な媒介物であること、および内皮細胞表面上でのへパラン硫酸 プロテオグリカンへの VEGFおよび bFGFの結合力 VEGFおよび bFGFの特異的受容 体への結合を調節することは周知である (Folkman J. et al.， Adv. Exp. Med. Biol. 31 3:355-64, 1992)。へパリン自体力 SVEGF依存的血管新生を促進することもまた、報告 されている (Folkman J. et al., Adv. Exp. Med. Biol. 313:355-64, 1992)。  [0097] Kallistatin is a heparin-binding serpin that inhibits tumor growth through suppression of angiogenesis (Miao R.Q. et al., Blood 100: 3245-52, 2002). VEGF and bFG F are potent mediators of angiogenesis, and the binding power of VEGF and bFGF to heparan sulfate proteoglycans on the surface of endothelial cells regulates the binding of VEGF and bFGF to specific receptors Are well known (Folkman J. et al., Adv. Exp. Med. Biol. 31 3: 355-64, 1992). Heparin itself has also been reported to promote SVEGF-dependent angiogenesis (Folkman J. et al., Adv. Exp. Med. Biol. 313: 355-64, 1992).
[0098] カリスタチンはへノリンに結合する能力により血管新生を阻害する力 この結合は V EGFがその受容体に結合するのに重要であり、したがってカリスタチンにより VEGF媒 介性血管新生が阻害される (Miao R.Q. et al., Am. J. Physiol. Cell Physiol. 284:C16 04-13, 2003)。へパリン結合セルピンであるアンチトロンビン (AT)の潜在型もまた抗 血管新生活性を有することが知られており (O'Reilly M.S. et al., Science 285:1926-8, 1999)、潜在型 ATが内皮細胞において、血管新生促進性 (proangiogenetic)プロテオ ダリカン、パールカンの発現を下方制御することが最近報告された (Zhang W. et al, B lood 103:1185-91, 2004)。カリスタチンおよび ATのように、 PCIもまたへパリン結合セ ルビンである。本発明において、本発明者らは PCIが MDA-231の増殖および転移を 阻害することを見出し、したがって血管新生に及ぼす PCIの効果を評価した。  [0098] Calistatin has the ability to inhibit angiogenesis through its ability to bind henoline. This binding is important for VEGF to bind to its receptor, and therefore, it inhibits VEGF-mediated angiogenesis ( Miao RQ et al., Am. J. Physiol. Cell Physiol. 284: C16 04-13, 2003). A latent form of antithrombin (AT), a heparin-binding serpin, is also known to have anti-angiogenic activity (O'Reilly MS et al., Science 285: 1926-8, 1999). It has recently been reported that AT down-regulates the expression of proangiogenetic proteolycan, perlecan, in endothelial cells (Zhang W. et al, Blood 103: 1185-91, 2004). Like calistatin and AT, PCI is also a heparin-binding serbin. In the present invention, the inventors have found that PCI inhibits MDA-231 proliferation and metastasis and thus evaluated the effect of PCI on angiogenesis.
[0099] まず、マトリゲルに取り込んだ原型 PCI発現 MDA-231細胞の、へパリン存在下にお ける VEGF誘導性血管新生に及ぼす効果を評価した。 VEGF誘導性血管新生は、原 型 PCI発現 MDA-231細胞を含むマトリゲルでは阻害された力 Mock発現 MDA-231 細胞を含むマトリゲルでは阻害されなかった。さらに、へパリンの存在下で VEGFによ り誘導される血管新生の阻害活性は、 R354APCI,または分解型 PCIの N末端断片発 現 MDA-231細胞を含むマトリゲルにおいても認められた。原型 PCI、 R354APCI、およ び degPCIの抗血管新生活性は、 CAMアツセィおよび内皮細胞の管形成により確認さ れた。これらのデータから、 PCIによる血管新生の阻害が、そのプロテアーゼ阻害活 性によって媒介されないことが示唆される。これらの知見は、 in vivo実験で観察され た腫瘍増殖および転移に対する原型 PCI、 R354APCUおよび degPCIの阻害活性と 一致する。 APCはそのプロテアーゼ活性を介して血管新生を促進することが、最近報 告された (Uchiba M. et al., Circ. Res. 95:34-41, 2004)。し力し、本発明では、 PCIが APCの活性を阻害することにより血管新生を抑制することは認められな力つた。 [0099] First, prototype PCI-expressing MDA-231 cells incorporated into Matrigel in the presence of heparin. We evaluated the effects on VEGF-induced angiogenesis. VEGF-induced angiogenesis was inhibited by Matrigel containing prototypical PCI-expressing MDA-231 cells but not by Matrigel containing Mock-expressing MDA-231 cells. Furthermore, the inhibitory activity of angiogenesis induced by VEGF in the presence of heparin was also observed in Matrigel containing MDA-231 cells expressing R354APCI, or N-terminal fragment of degraded PCI. The anti-angiogenic activity of prototype PCI, R354APCI, and degPCI was confirmed by CAM assembly and endothelial cell tube formation. These data suggest that inhibition of angiogenesis by PCI is not mediated by its protease inhibitory activity. These findings are consistent with the inhibitory activity of prototype PCI, R354APCU and degPCI on tumor growth and metastasis observed in in vivo experiments. APC has recently been reported to promote angiogenesis through its protease activity (Uchiba M. et al., Circ. Res. 95: 34-41, 2004). However, in the present invention, it was recognized that PCI inhibits angiogenesis by inhibiting the activity of APC.
[0100] 次に、原型 PCI発現 MDA-231細胞による血管新生の阻害活性に及ぼすへパリンの 効果を評価した。 PCI発現細胞を含むマトリゲルにおける血管新生は、大量のへパリ ン存在下でほぼ完全に消失した（データは示さず)。さらに、 R354APCIおよび degPCI 発現 MDA-231細胞を含むマトリゲルにぉ 、ても、血管新生阻害活性が認められた。 これらの知見から、 PCIはへパリンにおける VEGFの競合相手として作用すること、お よび PCIは内皮細胞表面上のへパリンまたはへパリン様グリコサミノダリカンへの結合 により血管新生を阻害することが示唆される。このデータは、阻害活性を欠くがへパリ ン結合活性を有する切断型 ATの N末端領域が抗血管新生活性を有すること (O'Reill y M.S. et al., Science 285:1926-8, 1999)、およびカリスタチンのへパリン結合部位変 異体に抗血管新生活性がないこと (Miao RQ et al., Blood 100:3245-52, 2002)を示す 以前の報告と一致する。 PCIのへパリン結合部位は、正に荷電した残基を含む、セル ピン分子の Hおよび Dヘリックス上に位置することが報告されている (Neese L.L. et al., Arch. Biochem. Biophys. 355:101—8, 1998)(Shirk R.A. et al., J. Biol. Chem. 269:28 690-5, 1994)。 [0100] Next, the effect of heparin on the angiogenesis inhibitory activity of prototype PCI-expressing MDA-231 cells was evaluated. Angiogenesis in Matrigel containing PCI-expressing cells disappeared almost completely in the presence of large amounts of heparin (data not shown). Furthermore, angiogenesis inhibitory activity was also observed in Matrigel containing R354APCI and degPCI expressing MDA-231 cells. These findings suggest that PCI acts as a VEGF competitor in heparin, and that PCI inhibits angiogenesis by binding to heparin or heparin-like glycosaminodarlicans on the surface of endothelial cells. Is done. This data indicates that the N-terminal region of truncated AT lacking inhibitory activity but having heparin binding activity has anti-angiogenic activity (O'Reill y MS et al., Science 285: 1926-8, 1999 ), And consistent with previous reports showing that the heparin binding site variant of calistatin has no anti-angiogenic activity (Miao RQ et al., Blood 100: 3245-52, 2002). The heparin binding site of PCI has been reported to be located on the H and D helices of the serpin molecule, containing positively charged residues (Neese LL et al., Arch. Biochem. Biophys. 355: 101-8, 1998) (Shirk RA et al., J. Biol. Chem. 269: 28 690-5, 1994).
[0101] 要するに、本発明は、 PCIが腫瘍増殖および転移を in vivoで阻害すること、および P CIの抗転移および抗増殖活性がそのプロテアーゼ阻害活性に依存しないことを実証 した。これらの知見から、 PCIが腎細胞癌および乳癌等の様々な腫瘍の治療に潜在 的に有用であることが示唆される。 [0101] In short, the present invention demonstrates that PCI inhibits tumor growth and metastasis in vivo, and that the anti-metastatic and anti-proliferative activity of PCI is independent of its protease inhibitory activity. did. These findings suggest that PCI is potentially useful for the treatment of various tumors such as renal cell carcinoma and breast cancer.
産業上の利用可能性 Industrial applicability
本発明によって、 PCIはそのプロテアーゼ阻害作用依存的に、癌の浸潤を抑制する ことが明らかにされた。したがって、プロテアーゼ阻害作用を有する PCI誘導体は、癌 の浸潤の抑制剤として有用である。一方、 PCIによる癌における血管新生阻害作用は 、 PCIのプロテアーゼ阻害作用に依存しな力つた。同様に PCIによる癌細胞の増殖阻 害作用も、 PCIのプロテアーゼ阻害作用に依存しないことが示された。したがって、プ 口テアーゼ阻害作用が低い、あるいは欠失した PCI誘導体は、血管新生の阻害剤並 びに癌細胞の増殖抑制剤として有用である。  According to the present invention, it has been clarified that PCI suppresses cancer invasion depending on its protease inhibitory action. Therefore, PCI derivatives having protease inhibitory action are useful as inhibitors of cancer invasion. On the other hand, the angiogenesis inhibitory effect in cancer by PCI was dependent on PCI protease inhibitory action. Similarly, the inhibition of proliferation of cancer cells by PCI was shown to be independent of the protease inhibitory action of PCI. Therefore, PCI derivatives with low or no protease inhibitory activity are useful as angiogenesis inhibitors and cancer cell growth inhibitors.
血管新生と細胞増殖は癌の悪性ィ匕における重要なメカニズムである。したがって、 これらのメカニズムに作用してそれを阻害する抗癌剤は、癌の悪性化の防止、あるい は悪性ィ匕した癌の治療に有用である。特に、プロテアーゼに対する阻害作用を持た ない PCI誘導体は、 PCIによるプロテアーゼ阻害作用に起因する副作用の軽減が期 待できる。  Angiogenesis and cell proliferation are important mechanisms in cancer malignancy. Therefore, anticancer agents that act on these mechanisms and inhibit them are useful for preventing cancer malignancy or treating malignant cancers. In particular, PCI derivatives that do not have an inhibitory effect on proteases can be expected to reduce the side effects caused by the protease inhibitory action by PCI.

Claims

請求の範囲 The scope of the claims
[I] プロテイン cインヒビターまたはその誘導体を有効成分として含有する抗癌剤。  [I] An anticancer agent containing a protein c inhibitor or a derivative thereof as an active ingredient.
[2] 癌の増殖、癌の転移、および血管新生力 選択された活性の少なくとも 1つを抑制す る請求項 1に記載の抗癌剤。  [2] The anticancer agent according to [1], which suppresses at least one of selected activities of cancer growth, cancer metastasis, and angiogenic potential.
[3] 癌が乳癌である請求項 1または 2に記載の抗癌剤。 [3] The anticancer agent according to claim 1 or 2, wherein the cancer is breast cancer.
[4] プロテイン Cインヒビターの誘導体のプロテアーゼ阻害作用がプロテイン Cインヒビタ 一よりも低い請求項 2に記載の抗癌剤。  4. The anticancer agent according to claim 2, wherein the protease inhibitory action of the protein C inhibitor derivative is lower than that of the protein C inhibitor.
[5] プロテイン Cインヒビターの誘導体力 プロテイン Cインヒビターのへパリン結合領域を 含む蛋白質である請求項 4に記載の抗癌剤。 [5] The anticancer agent according to claim 4, wherein the protein C inhibitor is a protein containing a heparin-binding region of a protein C inhibitor.
[6] プロテイン Cインヒビターの誘導体力 へパリンおよびへパリン様グリコサミノダルカン のいずれかまたは両方との結合能を有する蛋白質である請求項 5に記載の抗癌剤。 [6] The anticancer agent according to claim 5, wherein the protein C inhibitor is a protein having a binding ability to either or both of heparin and heparin-like glycosaminodalcan.
[7] 癌の浸潤を抑制する請求項 1に記載の抗癌剤。 7. The anticancer agent according to claim 1, which suppresses cancer invasion.
[8] プロテイン Cインヒビターの誘導体がプロテアーゼ阻害作用を有する請求項 7に記載 の抗癌剤。  8. The anticancer agent according to claim 7, wherein the protein C inhibitor derivative has a protease inhibitory action.
[9] プロテイン Cインヒビターまたはその誘導体を投与する工程を含む、癌の治療方法。  [9] A method for treating cancer, comprising a step of administering a protein C inhibitor or a derivative thereof.
[10] 癌の増殖、癌の転移、および血管新生力 選択された活性の少なくとも 1つが抑制さ れる、請求項 9に記載の方法。 [10] The method according to claim 9, wherein at least one of cancer growth, cancer metastasis, and angiogenic activity is suppressed.
[II] 癌が乳癌である請求項 9または 10に記載の方法。  [II] The method according to claim 9 or 10, wherein the cancer is breast cancer.
[12] プロテイン Cインヒビターの誘導体のプロテアーゼ阻害作用がプロテイン Cインヒビタ 一よりも低い、請求項 10に記載の方法。  12. The method according to claim 10, wherein the protease inhibitory action of the protein C inhibitor derivative is lower than that of the protein C inhibitor.
[13] プロテイン Cインヒビターの誘導体力 プロテイン Cインヒビターのへパリン結合領域を 含む蛋白質である、請求項 12に記載の方法。 [13] The method according to claim 12, wherein the protein C inhibitor is a protein containing a heparin-binding region of a protein C inhibitor.
[14] プロテイン Cインヒビターの誘導体力 へパリンおよびへパリン様グリコサミノダルカン のいずれかまたは両方との結合能を有する蛋白質である、請求項 13に記載の方法。 [14] The method according to claim 13, wherein the protein C inhibitor has a binding ability to one or both of heparin and heparin-like glycosaminodalkane.
[15] 癌の浸潤が抑制される、請求項 9に記載の方法。 [15] The method of claim 9, wherein cancer invasion is suppressed.
[16] プロテイン Cインヒビターの誘導体がプロテアーゼ阻害作用を有する、請求項 15に記 載の方法。  16. The method according to claim 15, wherein the protein C inhibitor derivative has a protease inhibitory action.
[17] 抗癌剤の製造のためのプロテイン Cインヒビターまたはその誘導体の使用。 [17] Use of a protein C inhibitor or a derivative thereof for the manufacture of an anticancer agent.
[18] 抗癌剤が、癌の増殖、癌の転移、および血管新生カゝら選択された活性の少なくとも 1 つを抑制する、請求項 17に記載の使用。 18. The use according to claim 17, wherein the anticancer agent inhibits at least one of selected activities such as cancer growth, cancer metastasis, and angiogenesis.
[19] 癌が乳癌である、請求項 17または 18に記載の使用。 [19] The use according to claim 17 or 18, wherein the cancer is breast cancer.
[20] プロテイン Cインヒビターの誘導体のプロテアーゼ阻害作用がプロテイン Cインヒビタ 一よりも低い、請求項 18に記載の使用。  [20] The use according to claim 18, wherein the protein C inhibitor has a protease inhibitory action lower than that of the protein C inhibitor.
[21] プロテイン Cインヒビターの誘導体力 プロテイン Cインヒビターのへパリン結合領域を 含む蛋白質である、請求項 20に記載の使用。 [21] The use according to claim 20, which is a protein containing a heparin-binding region of a protein C inhibitor.
[22] プロテイン Cインヒビターの誘導体力 へパリンおよびへパリン様グリコサミノダルカン のいずれかまたは両方との結合能を有する蛋白質である、請求項 21に記載の使用。 [22] The use according to claim 21, which is a protein having the ability to bind to either or both of heparin and heparin-like glycosaminodalcan.
[23] 抗癌剤が、癌の浸潤を抑制する、請求項 17に記載の使用。 [23] The use according to claim 17, wherein the anticancer agent suppresses cancer invasion.
[24] プロテイン Cインヒビターの誘導体がプロテアーゼ阻害作用を有する、請求項 23に記 載の使用。  [24] The use according to claim 23, wherein the derivative of the protein C inhibitor has a protease inhibitory action.
PCT/JP2006/308095 2005-04-18 2006-04-18 Anti-cancer agent comprising protein c inhibitor WO2006112451A1 (en)

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